Transgenic plants expressing cellulolytic enzymes

ABSTRACT

The invention provides novel methods of controlling gene expression in plastids, using an inducible, transactivator-mcdiated system, and plants comprising the novel expression systems. The present invention further describes the production of cellulose-degrading enzymes in plants via the application of genetic engineering techniques. Cellulase coding sequences are fused to promoters active in plants and transformed into the nuclear genome and the chloroplast genome. As cellulases may be toxic to plants, preferred promoters are those that are chemically-inducible. In this manner, expression of the cellulase genes transformed into plants may be chemically induced at an appropriate time. In addition, the expressed cellulases may be targeted to vacuoles or other organelles to alleviate toxicity problems. The present invention finds utility in any industrial process requiring a plentiful supply of cellulases, but particularly finds utility in the conversion of cellulosic biomass to ethanol.

FIELD OF THE INVENTION

[0001] The invention relates to the control of gene expression intransgenic plastids and to transgenic plants capable of expressingcellulose-degrading enzymes.

BACKGROUND OF THE INVENTION Industrial Uses for Cellulose-DegradingEnzymes

[0002] 1. Converting Biomass to Ethanol

[0003] The production of ethanol has received considerable attentionover the years as an octane booster, fuel extender, or neat liquid fuel.For example, in Brazil, up to 90% of new cars run on neat ethanol,whereas the remainder run on an ethanol/gasoline blend. In the UnitedStates, about 7% of all gasoline sold currently contains ethanol,usually a blend of 90% gasoline:10% ethanol. Fuel ethanol is currentlyproduced primarily from sugar cane in Brazil; however, in the UnitedStates, sugar prices are typically too high to make sugarcane attractiveas a feedstock for ethanol production. In the United States, fuelethanol is currently produced primarily from corn and other starch-richgrains. However, the production of one billion gallons of ethanol peryear corresponds to 400 million bushels of corn per year, which meansthat the existing corn ethanol industry is insufficient to supply thecurrent fuel market. In addition, corn ethanol is currently tooexpensive to cost-effectively compete with gasoline. To make asignificant impact on the transportation fuel market, ethanol needs abroader and cheaper resource base than industry currently has at itsdisposal. Technology for utilizing cellulosic biomass, for example wood,grass, and waste biomass from various commercial processes, as afeedstock could expand the resource base to accommodate most of the fuelmarket needs in the United States, because cellulosic biomass is cheapand plentiful.

[0004] The major components of terrestrial plants are two families ofsugar polymers, cellulose and hemicellulose. Cellulose fibers comprise4%-50% of the total dry weight of stems, roots, and leaves. These fibersare embedded in a matrix of hemicellulose and phenolic polymers.Cellulose is a polymer composed of six-carbon sugars, mostly glucose,linked by β-1,4 linkages. Hemicellulose is a polymer of sugars, but thetypes of sugars vary with the source of biomass. With the exception ofsoftwoods, the five-carbon sugar xylose is the predominant component inhemicellulose.

[0005] While all ethanol production ultimately involves fermentationprocesses from sugars, the technology for ethanol production fromcellulosic biomass is fundamentally different from ethanol productionfrom starchy food crops. While both require hydrolysis of the feedstock(starch or cellulose) into fermentable sugars, starch is easier tohydrolyze and enzymes that degrade starch, amylases, are relativelyinexpensive. In contrast, cellulose degrading enzymes or “cellulases”are currently less effective and more expensive. Hydrolysis ofcellulosic biomass to fermentable sugars can also occur though acidhydrolysis processes, which will not be discussed in detail. Cellulasesare a family of enzymes that work in concert to break down cellulose toits simple sugar components under much milder conditions compared toacid hydrolysis. In addition, these enzymes catalyze highly specificreactions and are required in much smaller quantities compared to acidhydrolysis reactions.

[0006] Hydrolysis of cellulose and starch produces glucose by thefollowing reaction:

n C₆H₁₀O₅+n H₂O→n C₆H₁₂O₆

[0007] After glucose is formed, fermentation thereof to ethanol proceedsby the following reaction:

C₆H₁₂O₆→2 CO₂+2 C₂H₅OH

[0008] For lignocellulosic biomass such as hardwoods, the hemicellulosicfraction must also be considered. For biomass predominantly containingthe five-carbon sugar xylose in the hemicellulose, the hydrolysisreaction proceeds as follows:

n C₅H₈O₄+n H₂O→n C₅H₁₀O₅

[0009] whereas the xylose produced is fermented to ethanol with thefollowing stoichiometry:

3 C₅H₁₀O₅→5 CO₂+5 C₂H₅OH

[0010] 2. Other Potential Uses for Cellulose-Degrading Enzymes

[0011] In addition to use in converting biomass to ethanol, cellulaseshave potential utility in other industrial processes, such as anyindustrial process that depends on a supply of fermentable sugars.Cellulases also have potential utility in the pulp and paper industryand in the textile industry to reduce the current dependency on acidhydrolysis, which is a major cause of water pollution.

[0012] In the animal feed industry, cellulases have utility as a feedadditive to aid the digestion of cellulosic material. Silage, forexample, can be made more digesible by the addition of cellulases orplants which express cellulases.

Characteristics of Cellulose-Degrading Enzymes

[0013] As stated above, cellulose and hemicellulose are the principalsources of fermentable sugars in lignocellulosic feedstocks; however,nature has designed woody tissue for effective resistance to microbialattack. A wide variety of organisms including bacteria and fungi possesscellulolytic activity. To be effective, cellulose-degradingmicroorganisms typically produce cellulase enzyme systems characterizedby multiple enzymatic activities that work synergistically to reducecellulose to cellobiose, and then to glucose. At least three differentenzymatic activities are required to accomplish this task.β-1,4-endoglucanases (EC 3.2.1.4, also called endocellulases) cleaveβ1,4-glycosidic linkages randomly along the cellulose chain.β-1,4-exoglucanases (EC 3.2.1.91, also called cellobiohydrolases, CBH)cleave cellobiose from either the reducing or the non-reducing end of acellulose chain. 1,4-β-D-glucosidases (EC 3.2.1.21, also calledcellobioses) hydrolyze aryl- and alkyl-β-D-glucosides.

[0014] Filamentous fungi are well known as a resource for industrialcellulases. However, this resource is generally regarded as tooexpensive for large scale industrial ethanol production. Some of themost prolific producers of extracellular cellulases are various strainsof Trichoderma reesei. By contrast, cellulases are typically produced bycelluloytic bacteria such as Thermomonospora fusca at much lower levelthan by filamentous fungi. However, cellulases from T. fusca and otherbacteria have been shown to have very high specific activities over abroad pH range and also have the desirable property of thermalstability. The T. fusca genes that encode cellulose-degrading enzymeshave been cloned and extensively characterized. (See, e.g., Collmer etal. (1983) Bio/Technology 1:594-601, hereby incorporated by reference;Ghangas et al. (1988) Appl. Environ. Microbiol. 54:2521-2526, herebyincorporated by reference; and Wilson (1992) Crit. Rev. Biotechnol.12:45-63, hereby incorporated by reference). In addition, the DNAsequences of a cellobiohydrolase gene and an endoglucanase gene from T.fusca have been determined (Jung et al. (1993) AppI. Environ. Microbiol.59:3032-3043, hereby incorporated by reference); and the DNA sequencesof three endoglucanase genes from T. fusca have also been determined(Lao et al. (1991) J. Bacteriol. 173:3397-3407, hereby incorporated byreference).

[0015] Efforts are currently being undertaken to utilize recombinantcellulase-producing bacterial or fungal hosts to produce variouscellulases for use in biomass-to-ethanol processes. Candidate cellulasesto be used in such recombinant systems are selected based on factorssuch as kinetics, temperature and pH tolerance, resistance to endproduct inhibition, and their synergistic effects. (See, for example,Thomas et al., “Initial Approaches to Artificial Cellulase Systems forConversion of Biomass to Ethanol”; Enzymatic Degradation of InsolubleCarbohydrates, J. N. Saddler and M. H. Penner, eds., ACS SymposiumSeries 618:208-36, 1995, American Chemical Society, Washington, D.C.,hereby incorporated by reference in its entirety). Examples ofheterologous expression of endoglucanases, exoglucanases, andβ-D-glucosidases in E. coli, Bacillus subtilis, and Streptomyceslividans have been reported (Lejeune et al., Biosynthesis andBiodegradation of Cellulose; Haigler, C. H.; Weimer, P. J., Eds.; MarcelDekker: New York, NY, 1990; pp. 623-671). In addition, the expression ofa B. subtilis endoglucanase and a C. fimi β-D-glucosidase in E. coli hasbeen demonstrated (Yoo et al. (1992) Biotechnol. Lett. 14:77-82).

[0016] While there is ongoing research to develop a multiple-geneexpression system in a suitable host that produces high levels ofendoglucanase, exoglucanase, and β-D-glucosidase activities in optimalproportions for the degradation of cellulosic biomass, the end result ofthis research will be simply be an improved bioreactor for producinglarge quantities of highly active cellulases for use in conventionalbiomass-to-ethanol processes as well as other industrial applications.Thus, this research is limited by the conventional problems inherentwith all such fermentation processes, including the fact that biomass isnaturally resistant to external enzymatic attack.

[0017] Current approaches to this problem are limited to usingrecombinant hosts that will not themselves be harmed by theirgenetically-engineered production of cellulose-degrading enzymes. Forexample, it would be expected that only hosts that do not themselvesinclude cellulose would be suitable for use in such bioreactors. Plants,therefore, would not be expected to be suitable hosts for recombinantcellulase genes. Transforming a plant to produce high levels ofcellulase is counterintuitive and presents special technicaldifficulties. To overcome these difficulties, it was necessary todevelop new expression systems, allowing for very high levels ofexpression, preferably under tight regulation to prevent damage to theplant during its development. These novel expression systems would alsohave applications beyond cellulase production.

SUMMARY OF THE INVENTION

[0018] In a first embodiment, the present invention addresses the needfor a plentiful, inexpensive source of cellulose-degrading enzymes forsuch industries as the fuel ethanol production industry, cattle feedindustry, and the paper and textile industries. Accordingly, the presentinvention provides for the production of cellulose-degrading enzymes inplants via the application of genetic engineering techniques. Higherplants make promising candidates for use as in vivo bioreactors forcellulase production. They have high biomass yields, production iseasily scaled up and does not require aseptic conditions, and complexpost-translational modification of plant-synthesized proteins iscommonplace. Moreover, levels of transgene-encoded proteins in plantsmay exceed 1% of total protein content. However, as plants depend oncellulose for structural integrity, it would be expected thatcellulose-degrading enzymes would be toxic to plants. Accordingly,cellulase genes represent an ideal target for technology relating to thechemical induction of gene expression and the targeting of gene productsto cell storage structures.

[0019] In the present invention, cellulase coding sequences are fused topromoters active in plants and transformed into the nuclear genome orthe plastid genome. Cellulases that may be expressed in plants accordingto the present invention include, but are not limited to,endoglucanases, exoglucanases, and β-D-glucosidases, preferably derivedfrom non-plant sources such as microorganisms (e.g. bacteria or fungi).A preferred promoter is the chemically-inducible tobacco PR-1a promoter;however, in certain situations, constitutive promoters such as the CaMV35S promoter may be used as well. With a chemically inducible promoter,expression of the cellulase genes transformed into plants may beactivated at an appropriate time by foliar application of a chemicalinducer.

[0020] Where plastid transformation is used, vectors are suitablyconstructed using a phage promoter, such as the phage T7 gene 10promoter, the transcriptional activation of which is dependent upon anRNA polymerase such as the phage T7 RNA polymerase. In one case, plastidtransformation vectors containing a phage promoter fused to a cellulasegene are transformed into the chloroplast genome. The resulting line iscrossed to a transgenic line containing a nuclear coding region for aphage RNA polymerase supplemented with a chloroplast-targeting sequenceand operably linked to a constitutive promoter such as the CAMV 35Spromoter, resulting in constitutive cellulase expression in thechloroplasts of plants resulting from this cross. Chloroplast expressionhas the advantage that the cellulase is less damaging to the plastid asit contains little or no cellulose.

[0021] In addition to using chemically-inducible promoters, theexpressed cellulases may be targeted to certain organelles such asvacuoles to alleviate toxicity problems. For vacuole-targeted expressionof cellulases, plants are transfonned with vectors that include avacuolar targeting sequence such as that from a tobacco chitinase gene.In this case, the expressed cellulases will be stored in the vacuoleswhere they will not be able to degrade cellulose and harm the plant.

[0022] The invention thus provides:

[0023] A plant which expresses a cellulose-degrading enzyme, e.g. acellulose degrading enzyme not naturally expressed in plants, forexample a plant comprising a heterologous DNA sequence coding for acellulose degrading enzyme stably integrated into its nuclear or plastidDNA, preferably under control of an inducible promoter, e.g., awound-inducible or chemically-inducible promoter, for example eitheroperably linked to the inducible promoter or under control oftransactivator-regulated promoter wherein the correspondingtransactivator is under control of the inducible promoter;

[0024] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag with instructions for use.

[0025] The invention further provides:

[0026] A method for producing a cellulose-degrading enzyme comprisingcultivating a cellulase-expressing plant;

[0027] a method for producing ethanol comprising fermenting acellulase-expressing plant; and

[0028] a method for enhancing the digestibility of animal feed, e.g.,silage, comprising adding a cellulase-expressing plant to the feed mix.

[0029] These methods may further comprise enhancing cellulosedegradation by combining two or more different cellulose degradingenzymes, e.g., enzymes acting at different stages in the cellulosebiodegradation pathway, e.g., in synergistically active combination,either by expressing said enzymes in a single plant or by combining twoor more plants each expressing a different cellulose degrading enzyme.

[0030] The invention further provides:

[0031] A plant expressible expression cassette comprising a codingregion for a cellulose-degrading enzyme, preferably under control of aninducible promoter, e.g., a wound inducible or chemically induciblepromoter; for example a plastid expressible expression cassettecomprising a promoter, e.g., a transactivator-mediated promoterregulated by a nuclear transactivator (e.g., the T7 promoter when thetransactivator is T7 RNA polymerase the expression of which isoptionally under control of an inducible promoter), and operably linkedto coding region for a cellulose-degrading enzyme;

[0032] a vector comprising such a plant expressible expression cassette;and

[0033] a plant transformed with such a vector, or a transgenic plantwhich comprises in its genome, e.g., its plasstid genome, such a plantexpressible expression cassette.

[0034] In a further embodiment, the present invention encompasses anovel system of plastid expression, wherein the gene expressed in theplastid is under control of a transactivator-regulated promoter, and thegene for the transactivator is in the nuclear DNA, under control of aninducible promoter. For example, plastid transformation vectors aretypically constructed using a phage promoter, such as the phage T7 gene10 promoter, the transcriptional activation of which is dependent uponan RNA polymerase such as the phage T7 RNA polymerase. The resultingline is crossed to a transgenic line containing a nuclear coding regionfor a phage RNA polymerase supplemented with a chloroplast-targetingsequence and operably linked to a chemically inducible promoter such asthe tobacco PR-1a promoter. Expression of the gene of interest in thechloroplasts of plants resulting from this cross is then activated byfoliar application of a chemical inducer. The novel, inducibletransactivator-mediated plastid expression system described herein isshown to be tightly regulatable, with no detectable expression prior toinduction and exceptionally high expression and accumulation of proteinfollowing induction.

[0035] The invention thus additionally provides:

[0036] A plant expressible expression cassette comprising an induciblepromoter, e.g., a wound-inducible or chemically-inducible promoter, forexample the tobacco PR-1a promoter, operably linked to a DNA sequencecoding for a transactivator (preferably a transactivator not naturallyoccurring in plants, preferably a RNA polymerase or DNA binding protein,e.g., T7 RNA polymerase), said transactivator being fused to a plastidtargeting sequence, e.g., a chloroplast targeting sequence;

[0037] a vector comprising such a plant expressible cassette; and

[0038] a plant transformed with such a vector or a transgenic plant thegenome of which comprises such a plant expressible expression cassette.

[0039] The invention furthermore provides:

[0040] A plant comprising

[0041] a heterologous nuclear expression cassette comprising aninducible promoter, e.g., a wound-inducible or chemically-induciblepromoter, for example the tobacco PR-1a promoter, operably linked to aDNA sequence coding for a transactivator (preferably a transactivatornot naturally occurring in plants, preferably a RNA polymerase or DNAbinding protein, e.g., T7 RNA polymerase), said transactivator beingoptionally fused to a plastid targeting sequence, e.g., a chloroplasttargeting sequence (e.g., a plant expressible expression cassette asdescribed above), and

[0042] a heterologous plastid expression cassette comprising atransactivator-mediated promoter regulated by the transactivator (e.g.,the T7 promoter when the transactivator is T7 RNA polymerase) andoperably linked to a DNA sequence of interest, e.g., coding for aprotein of interest (e.g., an enzyme, a carbohydrate degrading enzyme,for example GUS or a cellulose-degrading enzyme) or for a functional RNAof interest (e.g., antisense RNA);

[0043] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag or other container with instructions for use.

[0044] The invention also comprises:

[0045] A method of producing a plant as described above comprising

[0046] pollinating a plant comprising a heterologous plastid expressioncassette comprising a transactivator-mediated promoter regulated andoperably linked to a DNA sequence coding for a protein of interest

[0047] with pollen from a plant comprising a heterologous nuclearexpression cassette comprising an inducible promoter operably linked toa DNA sequence coding for a transactivator capable of regulating saidtransactivator-mediated promoter;

[0048] recovering seed from the plant thus pollinated; and

[0049] cultivating a plant as described above from said seed.

DEFINITIONS

[0050] “Cellulose-degrading enzymes” are described herein and includecellulases, cellobiohydrolases, cellobioses and other enzymes involvedin breaking down cellulose and hemicellulose into simple sugars such asglucose and xylose. Preferably, the cellulose-degrading enzyme used inthe present invention are of non-plant origin, e.g., of microbialorigin, preferably of bacterial origin, for example from a bacteria ofthe genus Thermomonospora, e.g., from T. fusca.

[0051] “Expression cassette” as used herein means a DNA sequence capableof directing expression of a gene in plant cells, comprising a promoteroperably linked to a coding region of interest which is operably linkedto a termination region. The coding region usually codes for a proteinof interest but may also code for a functional RNA of interest, forexample antisense RNA or a nontranslated RNA that, in the sense orantisense direction, inhibits expression of a particular gene, e.g.,antisense RNA. The gene may be chimeric, meaning that at least onecomponent of the gene is heterologous with respect to at least one othercomponent of the gene. The gene may also be one which is naturallyoccurring but has been obtained in a recombinant form useful for genetictransformation of a plant. Typically, however, the expression cassetteis heterologous with respect to the host, i.e., the particular DNAsequence of the expression cassette does not occur naturally in the hostcell and must have been introduced into the host cell or an ancestor ofthe host cell by a transformation event. A “nuclear expression cassette”is an expression cassette which is integrated into the nuclear DNA ofthe host. A “plastid expression cassette” is an expression cassettewhich is integrated into the plastid DNA of the host. A plastidexpression cassette as described herein may optionally comprise apolycistronic operon containing two or more cistronic coding sequencesof interest under control of a single promoter, e.g., atransactivator-mediated promoter, e.g., wherein one of the codingsequences of interest encodes an antisense mRNA which inhibitsexpression of clpP or other plastid protease, thereby enhancingaccumulation of protein expressed the the other coding sequence orsequences of interest.

[0052] “Heterologous” as used herein means “of different naturalorigin”. For example, if a plant is transformed with a gene derived fromanother organism, particularly from another species, that gene isheterologous with respect to that plant and also with respect todescendants of the plant which carry that gene.

[0053] “Homoplastidic” refers to a plant, plant tissue or plant cellwherein all of the plastids are genetically identical. This is thenormal state in a plant when the plastids have not been transformed,mutated, or otherwise genetically altered. In different tissues orstages of development, the plastids may take different forms, e.g.,chloroplasts, proplastids, etioplasts, amyloplasts, chromoplasts, and soforth.

[0054] An “inducible promoter” is a promoter which initiatestranscription only when the plant is exposed to some particular externalstimulus, as distinguished from constitutive promoters or promotersspecific to a specific tissue or organ or stage of development.Particularly preferred for the present invention arechemically-inducible promoters and wound-inducible promoters. Chemicallyinducible promoters include plant-derived promoters, such as thepromoters in the systemic acquired resistance pathway, for example thePR promoters, e.g., the PR-1, PR-2, PR-3, PR4, and PR-5 promoters,especially the tobacco PR-1a promoter and the Arabidopsis PR-1 promoter,which initiate transcription when the plant is exposed to BTH andrelated chemicals. See U.S. Pat. No. 5,614,395, incorporated herein byreference, and U.S. Provisional Application No. 60/027,228, incorporatedherein by reference. Chemically-inducible promoters also includereceptor-mediated systems, e.g., those derived from other organisms,such as steroid-dependent gene expression, copper-dependent geneexpression, tetracycline-dependent gene expression, and particularly theexpression system utilizing the USP receptor from Drosophila mediated byjuvenile growth hormone and its agonists, described in PCT/EP96/04224,incorporated herein by reference, as well as systems utilizingcombinations of receptors, e.g., as described in PCT/EP96/00686,incorporated herein by reference. Wound inducible promoters includepromoters for proteinase inhibitors, e.g., the proteinase inhibitor IIpromoter from potato, and other plant-derived promoters involved in thewound response pathway, such as promoters for polyphenyl oxidases, LAPand TD. See generally, C. Gatz, “Chemical Control of Gene Expression”,Annu. Rev. Plant Physiol. Plant Mol. Biol. (1997) 48: 89-108, thecontents of which are incorporated herein by reference.

[0055] A “plant” refers to any plant or part of a plant at any stage ofdevelopment. In some embodiments of the invention, the plants may belethally wounded to induce expression or may be induced to expresslethal levels of a desired protein, and so the term “plant” as usedherein is specifically intended to encompass plants and plant materialwhich have been seriously damaged or killed, as well as viable plants,cuttings, cell or tissue cultures, and seeds.

[0056] A “transactivator” is a protein which, by itself or incombination with one or more additional proteins, is capable of causingtranscription of a coding region under control of a correspondingtransactivator-mediated promoter. Examples of transactivator systemsinclude phage T7 gene 10 promoter, the transcriptional activation ofwhich is dependent upon a specific RNA polymerase such as the phage T7RNA polymerase. The transactivator is typically an RNA polymerase or DNAbinding protein capable of interacting with a particular promoter toinitiate transcription, either by activating the promoter directly or byinactivating a repressor gene, e.g., by suppressing expression oraccumulation of a repressor protein. The DNA binding protein may be achimeric protein comprising a binding region (e.g., the GALA bindingregion) linked to an appropriate transcriptional activator domain. Sometransactivator systems may have multiple transactivators, for examplepromoters which require not only a polymerase but also a specificsubunit (sigma factor) for promotor recognition, DNA binding, ortranscriptional activation. The transactivator is preferablyheterologous with respect to the plant.

DESCRIPTION OF THE FIGURES

[0057]FIG. 1 is a schematic description of chimeric gene constructsdescribed in the Examples for cellulase expression in plants. Hatchedboxes represent the E5 gene signal sequence and closed boxes representthe vacuolar targeting sequence from a tobacco chitinase gene. Tn codesfor nos termination sequences and Ttml for tml termination sequences.

[0058]FIG. 2 depicts plastid transformation vectors described in SectionC of the Examples.

DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention addresses the need for a plentiful,inexpensive source of cellulose-degrading enzymes for such industries asthe fuel ethanol production industry, cattle feed industry, and thepaper and textile industries by replacing the conventional industrialcellulases produced by fungi with cellulases produced in plants. Bygenetically engineering plants to produce their own cellulases, externalapplication of cellulases for cellulose degradation will be unnecessary.For example, lignocellulosic biomass destined to become ethanol couldserve as its own source of cellulase by utilizing the present invention.In fact, transgenic plants according to the present invention would notnecessarily have to comprise all of the feedstock in a bioreactor;rather, they could be used in conjunction with non-transforrnedcellulosic feedstock, whereby the cellulases produced by the transgenicplants would degrade the cellulose of all the feedstock, including thenon-transgenic feedstock. Cellulose degradation processes usingtransgenic biomass produced according to the present invention can becarried out more inexpensively, easily, and more environmentally safethan can conventional methods.

[0060] The feedstock could be any type of lignocelluosic material suchas high-biomass plants grown specifically for use as a source of biomassor waste portions of plants grown primarily for other purposes, such asstems and leaves of crop plants. Plants transformed with cellulase genesmay be transformed with constructs that provide constitutive expressionof cellulases if the particular plants can survive their own productionof cellulases. If a particular type of plant experiences undue toxicityproblems from the constitutive expression of cellulases, then the plantis preferably transformed with constructs that allow cellulaseproduction only when desired. For example, with chemically induciblecellulase constructs, one chemically induces cellulase expression justbefore harvesting plants so that just as the plants are being killed bytheir own production of cellulases, they are harvested anyway. Planttissue is then crushed, ground, or chopped to release the cellulasesthen added to a bioreactor in which the lignocellulosic biomass would bedegraded into simple sugars by the action of the cellulases expressed inthe transgenic plants.

[0061] The chimeric genes constructed according to the present inventionmay be transformed into any suitable plant tissue. As used inconjunction with the present invention, the term “plant tissue”includes, but is not limited to, whole plants, plant cells, plantorgans, plant seeds, protoplasts, callus, cell cultures, and any groupsof plant cells organized into structural and/or functional units. Plantstransformed in accordance with the present invention may be monocots ordicots and include, but are not limited to, maize, wheat, barley, rye,sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower,broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper,celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon,plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry,blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato,sorghum, sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco,carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis,and woody plants such as coniferous and deciduous trees.

[0062] Once a desired gene has been transformed into a particular plantspecies, it may be propagated in that species or moved into othervarieties of the same species, particularly including commercialvarieties, using traditional breeding techniques. Alternatively, thecoding sequence for a desired protein, e.g., a cellulose-degradingenzyme, may be isolated, genetically engineered for optimal expressionand then transformed into the desired variety

[0063] Preferred cellulase genes to be transformed into plants accordingto the present invention include, but are not limited to, the T. fuscaE1 gene (GenBank accession number L20094) (Jung et al. (1993) AppI.Environ. Microbiol. 59:3032-3043); the T. fusca E2 gene (GenBankaccession number M73321) (Ghangas et al. (1988) Appl. Environ.Microbiol. 54, 2521-2526; Lao et al. (199 1) J. Bacteriol. 173,3397-3407); and the T. fusca E5 gene (GenBank accession number L01577)(Collmer and Wilson (1983) Biotechnology 1, 594-601; Lao et al. (1991)J. Bacteriol. 173, 3397-3407). However, other cellulase genes may betransformed into plants according to the present invention as well,including all of the cellulase genes disclosed in the followingreferences: Collmer et al. (1983) Bio/Technology 1:594-601; Ghangas etal. (1988) Appl. Environ. Microbiol. 54:2521-2526: Wilson (1992) Crit.Rev. Biotechnol. 12:45-63; Jung et al. (1993) Appl. Environ. Microbiol.59:3032-3043; Lao et al. (1991) J. Bacteriol. 173:3397-3407; and Thomaset al., “Initial Approaches to Artificial Cellulase Systems forConversion of Biomass to Ethanol”; Enzymatic Degradation of InsolubleCarbohydrates, J. N. Saddler and M. H. Penner, eds., ACS SymposiumSeries 618:208-36, 1995, American Chemical Society, Washington, D.C.These include, but are not limited to, endoglucanases, exoglucanases,and β-D-glucosidases derived from microorganisms such as bacteria andfungi.

Modification of Microbial Genes to Optimize Nuclear Expression in Plants

[0064] If desired, the cloned cellulase genes described in thisapplication can be modified for expression in transgenic plant hosts.For example, the transgenic expression in plants of genes derived frommicrobial sources may require the modification of those genes to achieveand optimize their expression in plants. In particular, bacterial ORFsthat encode separate enzymes but which are encoded by the sametranscript in the native microbe are best expressed in plants onseparate transcripts. To achieve this, each microbial ORF is isolatedindividually and cloned within a cassette which provides a plantpromoter sequence at the 5′ end of the ORF and a plant transcriptionalterminator at the 3′ end of the ORF. The isolated ORF sequencepreferably includes the initiating ATG codon and the terminating STOPcodon but may include additional sequence beyond the initiating ATG andthe STOP codon. In addition, the ORF may be truncated, but still retainthe required activity; for particularly long ORFs, truncated versionswhich retain activity may be preferable for expression in transgenicorganisms. By “plant promoter” and “plant transcriptional terminator” itis intended to mean promoters and transcriptional terminators whichoperate within plant cells. This includes promoters and transcriptionterminators which may be derived from non-plant sources such as viruses(an example is the Cauliflower Mosaic Virus).

[0065] In some cases, modification to the ORF coding sequences andadjacent sequence will not be required, in which case it is sufficientto isolate a fragment containing the ORF of interest and to insert itdownstream of a plant promoter. Preferably, however, as little adjacentmicrobial sequence should be left attached upstream of the ATG anddownstream of the STOP codon. In practice, such construction may dependon the availability of restriction sites.

[0066] In other cases, the expression of genes derived from microbialsources may provide problems in expression. These problems have beenwell characterized in the art and are particularly common with genesderived from certain sources such as Bacillus. The modification of suchgenes can be undertaken using techniques now well known in the art. Thefollowing problems are typical of those that may be encountered:

[0067] 1. Codon Usage

[0068] The preferred codon usage in plants differs from the preferredcodon usage in certain microorganisms. Comparison of the usage of codonswithin a cloned microbial ORF to usage in plant genes (and in particulargenes from the target plant) will enable an identification of the codonswithin the ORF which should preferably be changed. Typically plantevolution has tended towards a strong preference of the nucleotides Cand G in the third base position of monocotyledons, whereas dicotyledonsoften use the nucleotides A or T at this position. By modifying a geneto incorporate preferred codon usage for a particular target transgenicspecies, many of the problems described below for GC/AT content andillegitimate splicing will be overcome.

[0069] 2. GC/AT Content

[0070] Plant genes typically have a GC content of more than 35%. ORFsequences which are rich in A and T nucleotides can cause severalproblems in plants. Firstly, motifs of ATTTA are believed to causedestabilization of messages and are found at the 3′ end of manyshort-lived mRNAs. Secondly, the occurrence of polyadenylation signalssuch as AATAAA at inappropriate positions within the message is believedto cause premature truncation of transcription. In addition,monocotyledons may recognize AT-rich sequences as splice sites (seebelow).

[0071] 3. Sequences Adiacent to the Initiating Methionine

[0072] Plants differ from microorganisms in that their messages do notpossess a defined ribosome binding site. Rather, it is believed thatribosomes attach to the 5′ end of the message and scan for the firstavailable ATG at which to start translation. Nevertheless, it isbelieved that there is a preference for certain nucleotides adjacent tothe ATG and that expression of microbial genes can be enhanced by theinclusion of a eukaryotic consensus translation initiator at the ATG.Clontech (1993/1994 catalog, page 210) have suggested the sequenceGTCGACCATGGTC as a consensus translation initiator for the expression ofthe E. coli uidA gene in plants. Further, Joshi (NAR 15: 6643-6653(1987)) has compared many plant sequences adjacent to the ATG andsuggests the consensus TAAACAATGGCT. In situations where difficultiesare encountered in the expression of microbial ORFs in plants, inclusionof one of these sequences at the initiating ATG may improve translation.In such cases the last three nucleotides of the consensus may not beappropriate for inclusion in the modified sequence due to theirmodification of the second AA residue. Preferred sequences adjacent tothe initiating methionine may differ between different plant species. Asurvey of 14 maize genes located in the GenBank database provided thefollowing results: Position Before the Initiating ATG in 14 Maize Genes:−10 −9 −8 −7 −6 −5 −4 −3 −2 −1 C 3 8 4 6 2 5 6 0 10  7 T 3 0 3 4 3 2 1 11 0 A 2 3 1 4 3 2 3 7 2 3 G 6 3 6 0 6 5 4 6 1 5

[0073] This analysis can be done for the desired plant species intowhich the cellulase genes are being incorporated, and the sequenceadjacent to the ATG modified to incorporate the preferred nucleotides.

[0074] 4. Removal of Illegitimate Splice Sites

[0075] Genes cloned from non-plant sources and not optimized forexpression in plants may also contain motifs which may be recognized inplants as 5′ or 3′ splice sites, and be cleaved, thus generatingtruncated or deleted messages. These sites can be removed using thetechniques described in application 07/961,944, hereby incorporated byreference.

[0076] Techniques for the modification of coding sequences and adjacentsequences are well known in the art. In cases where the initialexpression of a microbial ORF is low and it is deemed appropriate tomake alterations to the sequence as described above, then theconstruction of synthetic genes can be accomplished according to methodswell known in the art. These are, for example, described in thepublished patent disclosures EP 0 385 962 (to Monsanto), EP 0 359 472(to Lubrizol) and WO 93/07278 (to Ciba-Geigy). In most cases it ispreferable to assay the expression of gene constructions using transientassay protocols (which are well known in the art) prior to theirtransfer to transgenic plants.

[0077] A major advantage of plastid transformation is that plastids aregenerally capable of expressing bacterial genes without substantialmodification. Coden adaptation, etc. as described above is not required,and plastids are capable of expressing multiple open reading framesunder control of a single promoter.

Construction of Plant Transformation Vectors

[0078] Numerous transformation vectors are available for planttransformation, and the genes of this invention can be used inconjunction with any such vectors. The selection of vector for use willdepend upon the preferred transformation technique and the targetspecies for transformation. For certain target species, differentantibiotic or herbicide selection markers may be preferred. Selectionmarkers used routinely in transformation include the nptII gene whichconfers resistance to kanamycin and related antibiotics (Messing &Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187(1983)), the bar gene which confers resistance to the herbicidephoSphInothricin (White et al., Nucl Acids Res 18: 1062 (1990), Spenceret al. Theor Appl Genet 79: 625-631(1990)), the hpt gene which confersresistance to the antibiotic hygromycin (Blochinger & Diggelmann, MolCell Biol 4: 2929-2931), and the dhfr gene, which confers resistance tomethatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)).

[0079] 1. Construction of Vectors Suitable for AgrobacteriumTransformation

[0080] Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) andpXYZ. Below the construction of two typical vectors is described.

[0081] Construction of pCIB200 and pCIB2001: The binary vectors pCIB200and pCIB2001 are used for the construction of recombinant vectors foruse with Agrobacterium and was constructed in the following manner.pTJS75kan was created by NarI digestion of pTJS75 (Schmidhauser &Helinski, J Bacteriol. 164: 446-455 (1985)) allowing excision of thetetracycline-resistance gene, followed by insertion of an AccI fragmentfrom pUC4K carrying an NPTII (Messing & Vierra, Gene 19: 259-268 (1982);Bevan et al., Nature 304: 184-187 (1983); McBride et al., PlantMolecular Biology 14: 266-276 (1990)). XhoI linkers were ligated to theEcoRV fragment of pCIB7 which contains the left and right T-DNA borders,a plant selectable nos/nptII chimeric gene and the pUC polylinker(Rothstein et al., Gene 53: 153-161 (1987)), and the XhoI-digestedfragment was cloned into SalI-digested pTJS75kan to create pCIB200 (seealso EP 0 332 104, example 19). pCIB200 contains the following uniquepolylinker restriction sites: EcoRI, SstI, KpnI, BglII, Xbal, and SalI.pCIB2001 is a derivative of pCIB200 which created by the insertion intothe polylinker of additional restriction sites. Unique restriction sitesin the polylinker of pCIB2001 are EcoRI, SstI, KpnI, BglII, XbaI, SalI,MluI, BcllI AvrII, ApaI, HpaI, and StuI. pCIB2001, in addition tocontaining these unique restriction sites also has plant and bacterialkanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

[0082] Construction of pCIB10 and Hygromycin Selection Derivativesthereof: The binary vector pCIB10 contains a gene encoding kanamycinresistance for selection in plants, T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al. (Gene 53: 153-161 (1987)).Various derivatives of pCIB10 have been constructed which incorporatethe gene for hygromycin B phosphotransferase described by Gritz et al.(Gene 25: 179-188 (1983)). These derivatives enable selection oftransgenic plant cells on hygromycin only (pCIB743), or hygromycin andkanamycin (pCIB715, pCIB717).

[0083] 2. Construction of Vectors Suitable for non-AgrobacteriumTransformation

[0084] Transformation without the use of Agrobacterium tumefacienscircumvents the requirement for T-DNA sequences in the chosentransformation vector and consequently vectors lacking these sequencescan be utilized in addition to vectors such as the ones described abovewhich contain T-DNA sequences. Transformation techniques which do notrely on Agrobacterium include transformation via particle bombardment,protoplast uptake (e.g. PEG and electroporation) and microinjection. Thechoice of vector depends largely on the preferred selection for thespecies being transformed. Below, the construction of some typicalvectors is described.

[0085] Construction of pCIB3064: pCIB3064 is a pUC-derived vectorsuitable for direct gene transfer techniques in combination withselection by the herbicide basta (or phosphinothricin). The plasmidpCIB246 comprises the CaMV 35S promoter in operational fusion to the E.coli GUS gene and the CaMV 35S transcriptional terminator and isdescribed in the PCT published application WO 93107278. The 35S promoterof this vector contains two ATG sequences 5′ of the start site. Thesesites were mutated using standard PCR techniques in such a way as toremove the ATGs and generate the restriction sites SspI and PvuII. Thenew restriction sites were 96 and 37 bp away from the unique SalI siteand 101 and 42 bp away from the actual start site. The resultantderivative of pCIB246 was designated pCIB3025. The GUS gene was thenexcised from pCIB3025 by digestion with SalI and SacI, the terminirendered blunt and religated to generate plasmid pCIB3060. The plasmidpJIT82 was obtained from the John Innes Centre, Norwich and the a 400 bpSmaI fragment containing the bar gene from Streptomycesviridochromogenes was excised and inserted into the HpaI site ofpCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). This generatedpCIB3064 which comprises the bar gene under the control of the CaMV 35Spromoter and terminator for herbicide selection, a gene fro ampicillinresistance (for selection in E. coli) and a polylinker with the uniquesites SphI, PstI, HindIII, and BamHI. This vector is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

[0086] Construction of pSOG19 and pSOG35: pSOG35 is a transformationvector which utilizes the E. coli gene dihydrofolate reductase (DHFR) asa selectable marker conferring resistance to methotrexate. PCR was usedto amplify the 35S promoter (˜800 bp), intron 6 from the maize AdhI gene(˜550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10.A 250 bp fragment encoding the E. coli dihydrofolate reductase type IIgene was also amplified by PCR and these two PCR fragments wereassembled with a SacI-PstI fragment from pBI221 (Clontech) whichcomprised the pUC19 vector backbone and the nopaline synthaseterminator. Assembly of these fragments generated pSOG19 which containsthe 35S promoter in fusion with the intron 6 sequence, the GUS leader,the DHFR gene and the nopaline synthase terminator. Replacement of theGUS leader in pSOG19 with the leader sequence from Maize ChloroticMottle Virus (MCMV) generated the vector pSOG35. pSOG19 and pSOG35 carrythe pUC gene for ampicillin resistance and have HindIII, SphI, PstI andEcoRI sites available for the cloning of foreign sequences.

Requirements for Construction of Plant Expression Cassettes

[0087] Gene sequences intended for expression in transgenic plants arefirstly assembled in expression cassettes behind a suitable promoter andupstream of a suitable transcription terminator. These expressioncassettes can then be easily transferred to the plant transformationvectors described above.

[0088] 1. Promoter Selection

[0089] The selection of promoter used in expression cassettes willdetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, meosphyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and this selection will reflect the desiredlocation of biosynthesis of the cellulase. Alternatively, the selectedpromoter may drive expression of the gene under a light-induced or othertemporally regulated promoter. A further (and preferred) alternative isthat the selected promoter be inducible by an external stimulus, e.g.,application of a specific chemical inducer or wounding. This wouldprovide the possibility of inducing cellulase transcription only whendesired.

[0090] 2. Transcriptional Terminators

[0091] A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and its correct polyadenylation.Appropriate transcriptional terminators and those which are known tofunction in plants and include the CaMV 35S terminator, the tmlterminator, the nopaline synthase terminator, thc pea rbcS E9terminator. These can be used in both monocoylyedons and dicotyledons.

[0092] 3. Sequences for the Enhancement or Regulation of Expression

[0093] Numerous sequences have been found to enhance gene expressionfrom within the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

[0094] Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize AdhI gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develep 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronze1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

[0095] A number of non-translated leader sequences derived from virusesare also known to enhance expression, and these are particularlyeffective in dicotyledonous cells. Specifically, leader sequences fromTobacco Mosaic Virus (TMV, the “Ω-sequence”), Maize Chlorotic MottleVirus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to beeffective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res.15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15; 65-79(1990)).

[0096] 4. Targeting of the Gene Product Within the Cell

[0097] Various mechanisms for targeting gene products are known to existin plants and the sequences controlling the functioning of thesemechanisms have been characterized in some detail. For example, thetargeting of gene products to the chloroplast is controlled by a signalsequence found at the aminoterminal end of various proteins and which iscleaved during chloroplast import yielding the mature protein (e.g.Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signalsequences can be fused to heterologous gene products to effect theimport of heterologous products into the chloroplast (van den Broeck etal. Nature 313: 358-363 (1985)). DNA encoding for appropriate signalsequences can be isolated from the 5′ end of the cDNAs encoding theRUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2protein and many other proteins which are known to be chloroplastlocalized.

[0098] Other gene products are localized to other organelles such as themitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol.13: 411-418 (1989)). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms formitochondria. Targeting to cellular protein bodies has been described byRogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

[0099] In addition, sequences have been characterized which cause thetargeting of gene products to other cell compartments. Aminoterminalsequences are responsible for targeting to the ER, the apoplast, andextracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2:769-783 (1990)). Additionally, aminoterminal sequences in conjunctionwith carboxyterninal sequences are responsible for vacuolar targeting ofgene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

[0100] By the fusion of the appropriate targeting sequences describedabove to transgene sequences of interest it is possible to direct thetransgene product to any organelle or cell compartment. For chloroplasttargeting, for example, the chloroplast signal sequence from the RUBISCOgene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused inframe to the aminoterminal ATG of the transgene. The signal sequenceselected should include the known cleavage site and the fusionconstructed should take into account any amino acids after the cleavagesite which are required for cleavage. In some cases this requirement maybe fulfilled by the addition of a small number of amino acids betweenthe cleavage site and the transgene ATG or alternatively replacement ofsome amino acids within the transgene sequence. Fusions constructed forchloroplast import can be tested for efficacy of chloroplast uptake byin vitro translation of in vitro transcribed constructions followed byin vitro chloroplast uptake using techniques described by (Bartlett etal. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology,Elsevier. pp 1081-1091 (1982); Wasmann et al. Mol. Gen. Genet. 205:446-453 (1986)). These construction techniques are well knownn the artand are equally applicable to mitochondria and peroxisomes. The choiceof targeting which may be required for cellulase genes will depend onthe cellular localization of the precursor required as the startingpoint for a given pathway. This will usually be cytosolic orchloroplastic, although it may is some cases be mitochondrial orperoxisomal.

[0101] The above-described mechanisms for cellular targeting can beutilized not only in conjunction with their cognate promoters, but alsoin conjunction with heterologous promoters so as to effect a specificcell targeting goal under the transcriptional regulation of a promoterwhich has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

Examples of Expression Cassette Construction

[0102] The present invention encompasses the expression of cellulasegenes under the regulation of any promoter that is expressible inplants, regardless of the origin of the promoter.

[0103] Furthermore, the invention encompasses the use of anyplant-expressible promoter in conjunction with any further sequencesrequired or selected for the expression of the cellulase gene. Suchsequences include, but are not restricted to, transcriptionalterminators, extraneous sequences to enhance expression (such as introns[e.g. Adh intron 1], viral sequences [e. g. TMV-Ω]), and sequencesintended for the targeting of the gene product to specific organellesand cell compartments.

[0104] 1. Constitutive Expression: the CaMV 35S Promoter

[0105] Construction of the plasmid pCGN1761 is described in thepublished patent application EP 0 392 225 (example 23). pCGN1761contains the “double” 35S promoter and the tml transcriptionalterminator with a unique EcoRI site between the promoter and theterminator and has a pUC-type backbone. A derivative of pCGN1761 wasconstructed which has a modified polylinker which includes NotI and XhoIsites in addition to the existing EcoRI site. This derivative wasdesignated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNAsequences or gene sequences (including microbial ORF sequences) withinits polylinker for the purposes of their expression under the control ofthe 35S promoter in transgenic plants. The entire 35S promoter-genesequence-tml terminator cassette of such a construction can be excisedby HindIII, SphI, SalI, and XbaI sites 5′ to the promoter and XbaI,BamHI and BglI sites 3′ to the terminator for transfer to transformationvectors such as those described above. Furthermore, the double 35Spromoter fragment can be removed by 5′ excision with HindIII, SphI,SalI, XbaI, or PstI, and 3′ excision with any of the polylinkerrestriction sites (EcoRI, NotI or XhoI) for replacement with anotherpromoter.

[0106] 2. Modification of pCGN1761ENX by Optimization of theTranslational Initiation Site

[0107] For any of the constructions described in this section,modifications around the cloning sites can be made by the introductionof sequences which may enhance translation. This is particularly usefulwhen genes derived from microorganisms are to be introduced into plantexpression cassettes as these genes may not contain sequences adjacentto their initiating methionine which may be suitable for the initiationof translation in plants. In cases where genes derived frommicroorganisms are to be cloned into plant expression cassettes at theirATG it may be useful to modify the site of their insertion to optimizetheir expression. Modification of pCGN1761ENX is described by way ofexample to incorporate one of several optimized sequences for plantexpression (e.g. Joshi, supra).

[0108] pCGN1761ENX is cleaved with SphI, treated with T4 DNA polymeraseand religated, thus destroying the SphI site located 5′ to the double35S promoter. This generates vector pCGN1761ENX/Sph-. pCGN1761ENX/Sph-is cleaved with EcoRI, and ligated to an annealed molecular adaptor ofthe sequence 5′-AATTCTAAAGCATGCCGATCGG-3′/5′-AATTCCGATCGGCATGCTTTA-3′.This generates the vector pCGNSENX which incorporates thequasi-optimized plant translational initiation sequence TAAA-C adjacentto the ATG which is itself part of an SphI site which is suitable forcloning heterologous genes at their initiating methionine. Downstream ofthe SphI site, the EcoRI, NotI, and XhoI sites are retained.

[0109] An alternative vector is constructed which utilizes an NcoI siteat the initiating ATG. This vector, designated pCGN1761NENX is made byinserting an annealed molecular adaptor of the sequence5′-AATTCTAAACCATGGCGATCGG-3′/5′-AATTCCGATCGCCATGGTTTA-3′ at thepCGN1761ENX EcoRI site. Thus, the vector includes the quasi-optimizedsequence TAAACC adjacent to the initiating ATG which is within the NcoIsite. Downstream sites are EcoRI, NotI, and XhoI. Prior to thismanipulation, however, the two NcoI sites in the pCGN1761ENX vector (atupstream positions of the 5′ 35S promoter unit) are destroyed usingsimilar techniques to those described above for SphI or alternativelyusing “inside-outside” PCR (Innes et al. PCR Protocols: A guide tomethods and applications. Academic Press, New York (1990). Thismanipulation can be assayed for any possible detrimental effect onexpression by insertion of any plant cDNA or reporter gene sequence intothe cloning site followed by routine expression analysis in plants.

[0110] 3. Expression under a Chemically Regulatable Promoter

[0111] This section describes the replacement of the double 35S promoterin pCGN1761ENX with any promoter of choice; by way of example, thechemically regulatable PR-1a promoter is described in U.S. Pat. No.5,614,395, which is hereby incorporated by reference in its entirety,and the chemically regulatable Arabidopsis PR-1 promoter is described inU.S. Provisional Application No. 60/027,228, incorporated herein byreference. The promoter of choice is preferably excised from its sourceby restriction enzymes, but can alternatively be PCR-amplified usingprimers which carry appropriate terminal restriction sites. ShouldPCR-amplification be undertaken, then the promoter should be resequencedto check for amplification errors after the cloning of the amplifiedpromoter in the target vector. The chemically regulatable tobacco PR-1apromoter is cleaved from plasmid pCIB1004 (see EP 0 332 104, example 21for construction) and transferred to plasmid pCGN1761ENX. pCIB1004 iscleaved with NcoI and the resultant 3′ overhang of the linearizedfragment is rendered blunt by treatment with T4 DNA polymerase. Thefragment is then cleaved with HindIII and the resultant PR-1a promotercontaining fragment is gel purified and cloned into pCGN1761ENX fromwhich the double 35S promoter has been removed. This is done by cleavagewith XhoI and blunting with T4 polymerase, followed by cleavage withHindIII and isolation of the larger vector-terminator containingfragment into which the pCIB1004 promoter fragment is cloned. Thisgenerates a pCGN1761ENX derivative with the PR-1a promoter and the tmlterminator and an intervening polylinker with unique EcoRI and NotIsites. Selected cellulase genes can be inserted into this vector, andthe fusion products (i.e. promoter-gene-terminator) can subsequently betransferred to any selected transformation vector, including thosedescribed in this application.

[0112] Various chemical regulators may be employed to induce expressionof the cellulase coding sequence in the plants transformed according tothe present invention. In the context of the instant disclosure,“chemical regulators” include chemicals known to be inducers for thePR-1a promoter in plants, or close derivatives thereof. A preferredgroup of regulators for the chemically inducible cellulase genes of thisinvention is based on the benzo-1,2,3-thiadiazole (BTH) structure andincludes, but is not limited to, the following types of compounds:benzo-1,2,3-thiadiazolecarboxylic acid,benzo-1,2,3-thiadiazolethiocarboxylic acid,cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazolecarboxylic acidamide, benzo-1,2,3-thiadiazolecarboxylic acid hydrazide,benzo-1,2,3-thiadiazole-7-carboxylic acid, benzo- I,2,3-thiadiazole-7-thiocarboxylic acid, 7-cyanobenzo- 1,2,3-thiadiazole,benzo- 1,2,3-thiadiazole-7-carboxylic acid amide,benzo-1,2,3-thiadiazole-7-carboxylic acid hydrazide, alkylbenzo-1,2,3-thiadiazolecarboxylate in which the alkyl group contains oneto six carbon atoms, methyl benzo-1,2,3-thiadiazole-7-carboxylate,n-propyl benzo-1,2,3-thiadiazole-7-carboxylate, benzylbenzo-1,2,3-thiadiazole-7-carboxylate,benzo-1,2,3-thiadiazole-7-carboxylic acid sec-butylhydrazide, andsuitable derivatives thereof. Other chemical inducers may include, forexample, benzoic acid, salicylic acid (SA), polyacrylic acid andsubstituted derivatives thereof; suitable substituents include loweralkyl, lower alkoxy, lower alkylthio, and halogen. Still another groupof regulators for the chemically inducible DNA sequences of thisinvention is based on the pyridine carboxylic acid structure, such asthe isonicotinic acid structure and preferably the haloisonicotinic acidstructure. Preferred are dichloroisonicotinic acids and derivativesthereof, for example the lower alkyl esters. Suitable regulators of thisclass of compounds are, for example, 2,6-dichloroisonicotinic acid(INA), and the lower alkyl esters thereof, especially the methyl ester.

[0113] 4. Constitutive Expression: the Actin Promoter

[0114] Several isoforms of actin are known to be expressed in most celltypes and consequently the actin promoter is a good choice for aconstitutive promoter. In particular, the promoter from the rice ActIgene has been cloned and characterized (McElroy et al. Plant Cell 2:163-171 (1990)). A 1.3 kb fragment of the promoter was found to containall the regulatory elements required for expression in rice protoplasts.Furthermore, numerous expression vectors based on the ActI promoter havebeen constructed specifically for use in monocotyledons (McElroy et al.Mol. Gen. Genet. 231: 150-160 (1991)). These incorporate the ActI-intron1, AdhI 5′ flanking sequence and AdHI-intron 1 (from the maize alcoholdehydrogenase gene) and sequence from the CaMV 35S promoter. Vectorsshowing highest expression were fusions of 35S and the ActI intron orthe ActI 5′ flanking sequence and the ActI intron. Optimization ofsequences around the initiating ATG (of the GUS reporter gene) alsoenhanced expression. The promoter expression cassettes described byMcElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easilymodified for the expression of cellulase genes and are particularlysuitable for use in monocotyledonous hosts. For example, promotercontaining fragments can be removed from the McElroy constructions andused to replace the double 35S promoter in pCGN1761ENX, which is thenavailable for the insertion or specific gene sequences. The fusion genesthus constructed can then be transferred to appropriate transformationvectors. In a separate report the rice ActI promoter with its firstintron has also been found to direct high expression in cultured barleycells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).

[0115] 5. Constitutive Expression: the Ubiquitin Promoter

[0116] Ubiquitin is another gene product known to accumulate in manycall types and its promoter has been cloned from several species for usein transgenic plants (e.g. sunflower—Binet et al. Plant Science 79:87-94 (1991), maize - Christensen et al. Plant Molec. Biol. 12: 619-632(1989)). The maize ubiquitin promoter has been developed in transgenicmonocot systems and its sequence and vectors constructed for monocottransformation are disclosed in the patent publication EP 0 342 926 (toLubrizol). Further, Taylor et al. (Plant Cell Rep. 12: 491495 (1993))describe a vector (pAHC25) which comprises the maize ubiquitin promoterand first intron and its high activity in cell suspensions of numerousmonocotyledons when introduced via microprojectile bombardment. Theubiquitin promoter is suitable for the expression of cellulase genes intransgenic plants, especially monocotyledons. Suitable vectors arederivatives of pAHC25 or any of the transformation vectors described inthis application, modified by the introduction of the appropriateubiquitin promoter and/or intron sequences.

[0117] 6. Root Specific Expression

[0118] Another pattern of expression for the cellulases of the instantinvention is root expression. A suitable root promoter is that describedby de Framond (FEBS 290: 103-106 (1991)) and also in the publishedpatent application EP 0 452 269 (to Ciba-Geigy). This promoter istransferred to a suitable vector such as pCGN1761ENX for the insertionof a cellulase gene and subsequent transfer of the entirepromoter-gene-terminator cassette to a transformation vector ofinterest.

[0119] 7. Wound Inducible Promoters

[0120] Wound-inducible promoters may also be suitable for the expressionof cellulase genes. Numerous such promoters have been described (e.g. Xuet al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792(1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner etal. Plant J. 3: 191-201 (1993)) and all are suitable for use with theinstant invention. Logemann et al. describe the 5′ upstream sequences ofthe dicotyledonous potato wunl gene. Xu et al. show that a woundinducible promoter from the dicotyledon potato (pin2) is active in themonocotyledon rice. Further, Rohrmeier & Lehle describe the cloning ofthe maize Wipl cDNA which is wound induced and which can be used toisolated the cognate promoter using standard techniques. Similarly,Firek et al. and Warner et al. have described a wound induced gene fromthe monocotyledon Asparagus officinalis which is expressed at localwound and pathogen invasion sites. Using cloning techniques well knownin the art, these promoters can be transferred to suitable vectors,fused to the cellulase genes of this invention, and used to expressthese genes at the sites of plant wounding.

[0121] 8. Pith-Preferred Expression

[0122] Patent Application WO 93/07278 (to Ciba-Geigy) describes theisolation of the maize trpA gene which is preferentially expressed inpith cells. The gene sequence and promoter extend up to −1726 from thestart of transcription are presented. Using standard molecularbiological techniques, this promoter or parts thereof, can betransferred to a vector such as pCGN1761 where it can replace the 35Spromoter and be used to drive the expression of a foreign gene in apith-preferred manner. In fact, fragments containing the pith-preferredpromoter or parts thereof can be transferred to any vector and modifiedfor utility in transgenic plants.

[0123] 9. Leaf-Specific Expression

[0124] A maize gene encoding phosphoenol carboxylase (PEPC) has beendescribed by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)).Using standard molecular biological techniques the promoter for thisgene can be used to drive the expression of any gene in a leaf-specificmanner in transgenic plants.

[0125] 10. Expression with Chloroplast Targeting

[0126] Chen & Jagendorf (J. Biol. Chem. 268: 2363-2367 (1993) havedescribed the successful use of a chloroplast transit peptide for importof a heterologous transgene. This peptide used is the transit peptidefrom the rbcS gene from Nicotiana plumbaginifolia (Poulsen et al. Mol.Gen. Genet. 205: 193-200 (1986)). Using the restriction enzymes DraI andSphI, or Tsp509I and SphI the DNA sequence encoding this transit peptidecan be excised from plasmid prbcS-8B and manipulated for use with any ofthe constructions described above. The DraI-SphI fragment extends from−58 relative to the initiating rbcS ATG to, and including, the firstamino acid (also a methionine) of the mature peptide immediately afterthe import cleavage site, whereas the Tsp509I-SphI fragment extends from−8 relative to the initiating rbcS ATG to, and including, the firstamino acid of the mature peptide. Thus, these fragments can beappropriately inserted into the polylinker of any chosen expressioncassette generating a transcriptional fusion to the untranslated leaderof the chosen promoter (e.g. 35S, PR-1a, actin, ubiquitin etc.), whilstenabling the insertion of a cellulase gene in correct fusion downstreamof the transit peptide. Constructions of this kind are routine in theart. For example, whereas the DraI end is already blunt, the 5′ Tsp509Isite may be rendered blunt by T4 polymerase treatment, or mayalternatively be ligated to a linker or adaptor sequence to facilitateits fusion to the chosen promoter. The 3′ SphI site may be maintained assuch, or may alternatively be ligated to adaptor of linker sequences tofacilitate its insertion into the chosen vector in such a way as to makeavailable appropriate restriction sites for the subsequent insertion ofa selected cellulase gene. Ideally the ATG of the SphI site ismaintained and comprises the first ATG of the selected cellulase gene.Chen & Jagendorf provide consensus sequences for ideal cleavage forchloroplast import, and in each case a methionine is preferred at thefirst position of the mature protein. At subsequent positions there ismore variation and the amino acid may not be so critical. In any case,fusion constructions can be assessed for efficiency of import in vitrousing the methods described by Bartlett et al. (In: Edelmann et al.(Eds.) Methods in Chloroplast Molecular Biology, Elsevier. pp 1081-1091(1982)) and Wasmann et al. (Mol. Gen. Genet. 205: 446453 (1986)).Typically the best approach may be to generate fusions using theselected cellulase gene with no modifications at the aminoterminus, andonly to incorporate modifications when it is apparent that such fusionsare not chloroplast imported at high efficiency, in which casemodifications may be made in accordance with the established literature(Chen & Jagendorf; Wasman et al.; Ko & Ko, J. Biol. Chem. 267:13910-13916 (1992)).

[0127] A preferred vector is constructed by transferring the DraI-SphItransit peptide encoding fragment from prbcS-8B to the cloning vectorpCGN1761ENX/Sph-. This plasmid is cleaved with EcoRI and the terminirendered blunt by treatment with T4 DNA polymerase. Plasmid prbcS-8B iscleaved with SphI and ligated to an annealed molecular adaptor of thesequence 5′-CCAGCTGGAATTCCG-3′/5′-CGGAATTCCAGCTGGCATG-3′. The resultantproduct is 5′-terminally phosphorylated by treatment with T4 kinase.Subsequent cleavage with DraI releases the transit peptide encodingfragment which is ligated into the blunt-end ex-FcoRI sites of themodified vector described above. Clones oriented with the 5′ end of theinsert adjacent to the 3′ end of the 35S promoter are identified bysequencing. These clones carry a DNA fusion of the 35S leader sequenceto the rbcS-8A promoter-transit peptide sequence extending from −58relative to the rbcS ATG to the ATG of the mature protein, and includingat that position a unique SphI site, and a newly created EcoRI site, aswell as the existing NotI and XhoI sites of pCGN1761ENX. This new vectoris designated pCGN1761/CT. DNA sequences are transferred to pCGN1761/CTin frame by amplification using PCR techniques and incorporation of anSphI, NSphI, or NlaIII site at the amplified ATG, which followingrestriction enzyme cleavage with the appropriate enzyme is ligated intoSphI-cleaved pCGN1761/CT. To facilitate construction, it may be requiredto change the second amino acid of cloned gene, however, in almost allcases the use of PCR together with standard site directed mutagenesiswill enable the construction of any desired sequence around the cleavagesite and first methionine of the mature protein.

[0128] A further preferred vector is constructed by replacing the double35S promoter of pCGN1761ENX with the BamHI-SphI fragment of prbcS-8Awhich contains the full-length light regulated rbcS-8A promoter from−1038 (relative to the transcriptional start site) up to the firstmethionine of the mature protein. The modified pCGN1761 with thedestroyed SphI site is cleaved with PstI and EcoRI and treated with T4DNA polymerase to render termini blunt. prbcS-8A is cleaved SphI andligated to the annealed molecular adaptor of the sequence describedabove. The resultant product is 5′-terminally phosphorylated bytreatment with T4 kinase. Subsequent cleavage with BamHI releases thepromoter-transit peptide containing fragment which is treated with T4DNA polymerase to render the BamHI terminus blunt. The promoter-transitpeptide fragment thus generated is cloned into the prepared pCGN1761ENXvector, generating a construction comprising the rbcS-8A promoter andtransit peptide with an SphI site located at the cleavage site forinsertion of heterologous genes. Further, downstream of the SphI sitethere are EcoRI (re-created), NotI, and XhoI cloning sites. Thisconstruction is designated pCGN1761 rbcS/CT.

[0129] Similar manipulations can be undertaken to utilize other GS2chloroplast transit peptide encoding sequences from other sources(monocotyledonous and dicotyledonous) and from other genes. In addition,similar procedures can be followed to achieve targeting to othersubcellular compartments such as mitochondria.

Transformation of Dicotyledons

[0130] Transformation techniques for dicotyledons are well known in theart and include Agrobacterium-based techniques and techniques which donot require Agrobacterium. Non-Agrobacterium techniques involve theuptake of exogenous genetic material directly by protoplasts or cells.This can be accomplished by PEG or electroporation mediated uptake,particle bombardment-mediated delivery, or microinjection. Examples ofthese techniques are described by Paszkowski et al., EMBO J 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

[0131] Agrobacterium-mediated transformation is a preferred techniquefor transformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species. Themany crop species which are routinely transformable by Agrobacteriuminclude tobacco, tomato, sunflower, cotton, oilseed rape, potato,soybean, alfalfa and poplar (EP 0 317 511 (cotton [1313]), EP 0 249 432(tomato, to Calgene), WO 87107299 (Brassica, to Calgene), U.S. Pat. No.4,795,855 (poplar)). Agrobacterium transformation typically involves thetransfer of the binary vector carrying the foreign DNA of interest (e.g.pCIB2000 or pCIB2001) to an appropriate Agrobacterium strain which maydepend of the complement of vir genes carried by the host Agrobacteriumstrain either on a co-resident Ti plasmid or chromosomally (e.g. strainCIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169(1993)). The transfer of the recombinant binary vector to Agrobacteriumis accomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höbfgen & Willmitzer, Nucl. Acids Res. 16: 9877(1988)).

[0132] Transformation of the target plant species by recombinantAgrobacterium usually involves co-cultivation of the Agrobacterium withexplants from the plant and follows protocols well known in the art.Transformed tissue is regenerated on selectable medium carrying theantibiotic or herbicide resistance marker present between the binaryplasmid T-DNA borders.

Transformation of Monocotyledons

[0133] Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complex vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

[0134] Patent Applications EP 0 292 435 ([1280/1281] to Ciba-Geigy), EP0 392 225 (to Ciba-Geigy) and WO 93/07278 (to Ciba-Geigy) describetechniques for the preparation of callus and protoplasts from an eliteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Frommet al. (Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, application WO 93/07278 (to Ciba-Geigy) and Koziel et al.(Biotechnology 11: 194-200 (1993)) describe techniques for thetransformation of elite inbred lines of maize by particle bombardment.This technique utilizes immature maize embryos of 1.5-2.5 mm lengthexcised from a maize ear 14-15 days after pollination and a PDS-1000HeBiolistics device for bombardment.

[0135] Transformation of rice can also be undertaken by direct genetransfer techniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al., Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).

[0136] Patent Application EP 0 332 581 (to Ciba-Geigy) describestechniques for the generation, transformation and regeneration ofPooideae protoplasts. These techniques allow the transformation ofDactylis and wheat. Furthermore, wheat transformation was been describedby Vasil et al. (Biotechnology 10: 667-674 (1992)) using particlebombardment into cells of type C long-term regenerable callus, and alsoby Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks et al.(Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment ofimmature embryos and immature embryo-derived callus. A preferredtechnique for wheat transformation, however, involves the transformationof wheat by particle bombardment of immature embryos and includes eithera high sucrose or a high maltose step prior to gene delivery. Prior tobombardment, any number of embryos (0.75-1 mm in length) are plated ontoMS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15:473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos whichis allowed to proceed in the dark. On the chosen day of bombardment,embryos are removed from the induction medium and placed onto theosmoticum (i.e. induction medium with sucrose or maltose added at thedesired concentration, typically 15%). The embryos are allowed toplasmolyze for 2-3 h and are then bombarded. Twenty embryos per targetplate is typical, although not critical. An appropriate gene-carryingplasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer sizegold particles using standard procedures. Each plate of embryos is shotwith the DuPont Biolistics® helium device using a burst pressure of˜1000 psi using a standard 80 mesh screen. After bombardment, theembryos are placed back into the dark to recover for about 24 h (stillon osmoticum). After 24 hrs, the embryos are removed from the osmoticumand placed back onto induction medium where they stay for about a monthbefore regeneration. Approximately one month later the embryo explantswith developing embryogenic callus are transferred to regenerationmedium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing theappropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2mg/l methotrexate in the case of pSOG35). After approximately one month,developed shoots are transferred to larger sterile containers known as“GA7s” which contained half-strength MS, 2% sucrose, and the sameconcentration of selection agent. Patent application No. 08/147,161describes methods for wheat transformation and is hereby incorporated byreference.

Chloroplast Transformation

[0137] Plastid transformation technology is extensively described inU.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, all of which arehereby expressly incorporated by reference in their entireties; in PCTapplication no. WO 95/16783, which is hereby incorporated by referencein its entirety; and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91, 7301-7305, which is also hereby incorporated by reference in itsentirety. The basic technique for chloroplast transformation involvesintroducing regions of cloned plastid DNA flanking a selectable markertogether with the gene of interest into a sutable target tissue, e.g.,using biolistics or protoplast transformation (e.g., calcium chloride orPEG mediated transformation). The 1 to 1.5 kb flanking regions, termedtargeting sequences, facilitate homologous recombination with theplastid genome and thus allow the replacement or modification ofspecific regions of the plastome. Initially, point mutations in thechloroplast 16S rRNA and rps12 genes conferring resistance tospectinomycin andlor streptomycin were utilized as selectable markersfor transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990)Proc. Natl. Acad. Sci. USA 87, 8526-8530, hereby incorporated byreference; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45,hereby incorporated by reference). This resulted in stable homoplasmictransformants at a frequency of approximately one per 100 bombardmentsof target leaves. The presence of cloning sites between these markersallowed creation of a plastid targeting vector for introduction offoreign genes (Staub, J. M., and Maliga, P. (1993) EMBO J. 12, 601-606,hereby incorporated by reference). Substantial increases intransformation frequency were obtained by replacement of the recessiverRNA or τ-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial aadA gene encoding the spectinomycin-detoxifyingenzyme aminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90, 913-917, hereby incorporated byreference). Previously, this marker had been used successfully forhigh-frequency transformation of the plastid genome of the green algaChlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. AcidsRes. 19, 4083-4089, hereby incorporated by reference). Other selectablemarkers useful for plastid transformation are known in the art andencompassesd within the scope of the invention. Typically, approximately15-20 cell division cycles following transformation are required toreach a homoplastidic state.

[0138] Plastid expression, in which genes are inserted by homologousrecombination into all of the several thousand copies of the circularplastid genome present in each plant cell, takes advantage of theenormous copy number advantage over nuclear-expressed genes to permitexpression levels that can readily exceed 10% of the total soluble plantprotein. However, such high expression levels may pose potentialviability problems, especially during early plant growth anddevelopment. Similar problems are posed by the expression of bioactiveenzymes or proteins that may be highly deleterious to the survival oftransgenic plants and henced if expressed constitutively may not beintroduced successfully into the plant genome. Thus, in one aspect, thepresent invention has coupled expression in the nuclear genome of achoroplast-targeted phage T7 RNA polymerase under control of thechemically inducible PR-1a promoter (U.S. Pat. No. 5,614,395incorporated by reference) of tobacco to a chloroplast reportertransgene regulated by T7 gene 10 promoter/terminator sequences. Forexample, when plastid transformants homoplasmic for the maternallyinherited uidA gene encoding the β-glucuronidase (GUS) reporter arepollinated by lines expressing the T7 polymerase in the nucleus, F1plants are obtained that carry both transgene constructs but do notexpress the GUS protein. Synthesis of large amounts of enzymaticallyactive GUS is triggered in plastids of these plants only after foliarapplication of the PR-1a inducer compoundbenzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH). As setforth below in Section C of the Examples, the present invention alsoentails the synthesis of large amounts of cellulose-degrading enzymesusing this chloroplast-targeted T7 RNA polymerase expression system.

[0139] The invention will be further described by reference to thefollowing detailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified.

EXAMPLES

[0140] Standard recombinant DNA and molecular cloning techniques usedhere are well known in the art and are described by J. Sambrook, E. F.Fritsch and T. Maniatis, Molecular Cloning: A Laboratory manual, ColdSpring Harbor laboratory, Cold Spring Harbor, N.Y. (1989) and by T. J.Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and byAusubel, F. M. et al., Current Protocols in Molecular Biology, pub. byGreene Publishing Assoc. and Wiley-Interscience (1987).

[0141] A. Expression of Cellulases in the Plant Cytosol

Example A1

[0142] Preparation of a chimeric gene containing the T. fusca E1cellulase coding sequence fused to the tobacco PR-1a promoter

[0143] Plasmid pGFE1 (Jung et al. (1993) Appl. Environ. Microbiol. 59,3032-3043) containing the T. fusca E1 gene (GenBank accession numberL20094), which codes for a protein with endoglucanase activity, was usedas the template for PCR with a left-to-right “top strand” primercomprising an ATG before the first codon of the mature E1 protein, thefirst 21 base pairs of the mature protein and a NcoI restriction site atthe newly created ATG (primer E11: GCG CCC ATG GAC GAA GTC AAC CAG ATTCGC) and a right-to-left “bottom strand” primer homologous to positions322 to 346 from the newly created ATG of the E1 gene (primer E12: CCAGTC GAC GTT GGA GGT GAA GAC). This PCR reaction was undertaken withAmpliTaq DNA polymerase according to the manufacturer's recommendations(Perkin Elmer/Roche, Branchburg, N.J.) for five cycles at 94° C. (30 s),40° C. (60 s), and 72° C. (30 s) followed by 25 cycles at 94° C. (30 s),55° C. (60 s) and 72° C. (30 s). This generated a product of 352 bpcontaining a NcoI site at its left end and a EcoRI site at its right endand comprised the 5′ end of the E1 gene without the signal sequence. Thefragment was gel purified using standard procedures, cleaved with NcoIand EcoRI (all restriction enzymes purchased from Promega, Madison, Wis.or New England Biolabs, Beverly, Mass.) and ligated into the NcoI andEcoRI sites of pTC191 (De La Fuente el al. (1994) Gene 139, 83-86) toobtain pE1.

[0144] Plasmid pGFE1 was then digested with EcoRI and ScaI. The 3.0 kblong EcoRI fragment containing the 3′ end of the E1 gene was gelpurified and ligated with pE1, which had previously been digested withEcoRI, to obtain pCTEI containing the entire E1 gene without a signalsequence. Plasmid pCTE1 was digested with NcoI and SacI. The 3.3 kb longfragment containing the E1 gene was gel purified and ligated into theNcoI and SacI sites of pJG203 between a 903 bp long tobacco PR-1apromoter and the nos gene termination signals (Uknes et al. (1993), ThePlant Cell 5,159-169, modified by removal of an additional SacI site,Joern Goerlach, notebook no. 2941, pp 4-9 and 13-15), yielding pTPRIE1containing the E1 gene fused to the tobacco PR-1a promoter (FIG. 1).

[0145] Plasmid pTPRIE1 was digested with XhoI and XbaI and the 4.5 kblong fragment containing the chimeric E1 gene construct was gel purifiedand ligated into the XhoI and XbaI sites of pBHYGM to obtain binaryvector pEGL101. pBHYGM is a modified pGPTV-Hyg vector (Becker et al.(1992) Plant Mol. Biol. 20, 1195-1197) produced by insertion of apolylinker containingBfrI/ApaI/ClaI/SmaI/BfrI/XbaI/SalI/PstI/SphI/HindIII restriction sitesinto the EcoRI and XbaI sites of pGPTV-Hyg.

Example A2

[0146] Preparation of a chimeric gene containing the T. fusca E2cellulase coding sequence fused to the tobacco PR-1a promoter

[0147] Plasmid pJT17 containing the T. fusca E2 gene (Ghangas et al.(1988) Appl. Environ. Microbiol. 54, 2521-2526; Lao et al. (1991) J.Bacteriol. 173, 3397-3407) (GenBank accession number M73321), whichcodes for a protein with cellobiohydrolase activity, was used as thetemplate for PCR with a left-to-right “top strand” primer comprising anATG before the last codon of the E2 signal sequence, the first 18 basepairs of the mature protein and a NcoI restriction site at the newlycreated ATG (primer E21: GCG CGC CAT GGC CAA TGA TTC TCC GTT CTA C)right-to-left “bottom strand” primer homologous to positions 310 to 334from the newly created ATG of the E2 gene (primer E22: GGG ACG GTT CTTCAG TCC GGC AGC). This PCR reaction was undertaken with AmpliTaq DNApolymerase according to the manufacturer's recommendations for fivecycles at 94° C. (30 s), 40° C. (60 s), and 72° C. (30 s) followed by 25cycles at 94° C. (30 s), 55° C. (60 s) and 72° C. (30 s). This generateda product of 341 bp containing a NcoI site at its left end and a EcoRIsite at its right end comprising the 5′ end of the E2 gene without asignal sequence. The fragment was gel purified using standardprocedures, cleaved with NcoI and EcoRI and ligated into the NcoI andEcoRI sites of pTC191 to obtain pE2.

[0148] Plasmid pJT17 was then digested with EcoRI and SacI. The 1.7 kblong fragment containing the 3′ end of the E2 gene was gel purified andligated with pE2, which had previously been digested with EcoRI andSacI, to obtain pCTE2 containing the entire E2 gene without a signalsequence. Plasmid pCTE2 was digested with NcoI and SacI and the 2.0 kblong fragment containing the E2 gene was gel purified and ligated intothe NcoI and SacI sites of pJG203, yielding pTPR1E2 containing the E2gene fused to a 903 bp long tobacco PR-1a promoter fragment (FIG. 1).

[0149] Plasmid pTPR1E2 was digested with XhoI and XbaI and the 2.9 kblong fragment containing the chimeric E2 gene construct was gel purifiedand ligated into the XhoI and XbaI sites of pBHYGM to construct pEGL102.

Example A3

[0150] Preparation of a chimeric gene containing the T. fusca E5cellulase coding sequence fused to the tobacco PR-1a promoter

[0151] Plasmid pD374, a modified version of pD370 (Collmer and Wilson(1983) Biotechnology 1, 594-601; Lao et al. (1991) J. Bacteriol. 173,3397-3407) containing the T. fusca E5 gene (GenBank accession numberL01577), which codes for a protein with endoglucanase activity, was usedas the template for PCR with a left-to-right “top strand” primercomprising an ATG before the first codon of the mature E5 protein, thefirst 21 base pairs of the mature protein and a NcoI restriction site atthe newly created ATG (primer E51: CGC CCA TGG CCG GTC TCA CCG CCA CAGTC) and a right-to-left “bottom strand” primer homologous to positions89 to 114 from the newly created ATG of the E5 gene (primer E52: GAC GACCTC CCA CTG GGA GAC GGT G). AmpliTaq DNA polymerase was used for PCRaccording to the manufacturer's recommendations for five cycles at 94°C. (30 s), 40° C. (60 s), and 72° C. (30 s) followed by 25 cycles at 94°C. (30 s), 55° C. (60 s) and 72° C. (30 s). This generated a product of119 bp containing a NcoI site at its left end and a XhoI site at itsright end and comprised the 5′ end of the E5 gene without a signalsequence. The fragment was gel purified, cleaved with NcoI and XhoI andligated into the NcoI and XhoI sites of pCIB4247 to obtain pCE5.pCIB4247 is a pUC19 derivative (Yanisch-Perron et al. (1985) Gene 33,103-119) containing a polylinker with NcoI, XhoI and EcoRI restrictionsites.

[0152] In order to reconstitute the entire E5 gene, a 1.4 kb longXhoI/PvuII fragment of pD374 containing the E5 gene 3′ end was subclonedinto the XhoI and EcoRV sites of pICEM19R+, a pUC19 derivativecontaining a polylinker with XhoI, EcoRV and EcoRI restriction sites,excised as a XhoI/EcoRI fragment and ligated into the XhoI and EcoRIsites of pCE5 to form pCTE5 containing the entire E5 gene. pCTE5 wasdigested with EcoRI, the protruding ends of the EcoRI site werefilled-in with DNA Polymerase I Klenow fragment (Promega, Madison, Wis.)and plasmid DNA was further digested with NcoI. The 1.5 kb long fragmentcontaining the E5 gene was gel purified and ligated into the NcoI andEcoICRI sites of pJG203, yielding pTPR1E5 containing the E5 gene fusedto a 903 bp long tobacco PR-1a promoter (FIG. 1).

[0153] Plasmid pTPR1E5 was digested with ApaI, XbaI and SacI and the 2.7kb long ApaI/XbaI fragment containing the chimeric E5 gene construct wasgel purified and ligated into the ApaI and XbaI sites of pBHYGM toconstruct pEGL105.

Example A4

[0154] Preparation of a chimeric gene containing the T. fusca E5cellulase coding sequence fused to the CaMV 35S promoter

[0155] A 1.5 kb long NcoI/EcoRI fragment of pCTE5 containing the E5 geneand whose protruding ends had been previously filled-in with Klenow DNAPolymerase was gel purified and ligated into the filled-in EcoRI site ofpCGN1761 between a duplicated CaMV 35S promoter (Kay el al. (1987)Science 236, 1299-1302) and the tml gene termination signals (Ream etal. (1983) Proc. Natl. Acad. Sci. USA 80, 1660-1664), resulting inp35SE5 (FIG. 1). A 4.6 kb long fragment of p35SE5 containing thechimeric gene was inserted into the XbaI site of pBHYGM to obtainpEGL355.

Example A5

[0156] Preparation of chimeric genes containing the T. fusca E1cellulase coding sequence fused to the CaMV 35S promoter

[0157] A 3.3 kb long NcoI (filled in)/SacI fragment of pCTE1 containingthe E1 gene is gel purified and ligated into the filled-in EcoRI site ofpCGN1761. The chimeric gene containing the E1 coding sequence fused tothe CaMV 35S promoter is inserted into the XbaI site of pBHYGM.

Example A6

[0158] Preparation of chimeric genes containing the T. fusca E2cellulase coding sequence fused to the CaMV 35S promoter

[0159] A 2.0 kb long NcoI (filled in)/SacI fragment of pCTE2 containingthe E2 gene is gel purified and ligated into the filled-in EcoRI site ofpCGN1761. The chimeric gene containing the E2 coding sequence fused tothe CaMV 35S promoter is inserted into the XbaI site of pBHYGM.

Example A7

[0160] Transformation of tobacco leaf discs by A. tumefaciens

[0161] The binary vector constructs pEGL101, pEGL102, pEGL105, andpEGL355 were transformed into A. tumefaciens strain GV3101 (Bechtold, N.et al. (1993), CR Acad. Sci. Paris, Sciences de la vie, 316:1194-1199)by electroporation (Dower, W. J. (1987), Plant Mol. Biol. Reporter 1:5).The same procedure is used for transformation of tobacco with otherconstructs containing chimeric cellulase genes.

[0162] Leaf discs of Nicotiana tabacum cv ‘Xanthi nc’ and of transgenicline “NahG” overexpressing a salicylate hydroxylase gene (Gaffney et al.(1993) Science 261: 754-756) were cocultivated with Agrobacterium clonescontaining the above mentioned constructs (Horsch et al. (1985), Science227: 1229-1231) and transformants were selected for resistance to 50μg/ml hygromycin B. Approximately 50 independent hygromycin lines (T0lines) for each construct were selected and rooted on hormone-freemedium.

Example A8

[0163] Transformation of maize

[0164] Maize transformation by particle bombardment of immature embryosis performed as described by Koziel et al. (Biotechnology 11, 194-200,1993).

Example A9

[0165] Transformation of wheat

[0166] Transformation of immature wheat embryos and immatureembryo-derived callus using particle bombardment is performed asdescribed by Vasil et. al. (Biotechnology 11: 1553-1558,1993) and Weekset. al. (Plant Physiology 102: 1077-1084, 1993).

Example A10

[0167] Selection of transgenic lines with inducible cellulase geneexpression

[0168] For each transgenic line, duplicate leaf punches of approximately2-3 cm² were incubated for 2 days in 3 ml ofbenzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH, 5.6mg/10 ml) or sterile distilled water under ca. 300 μmol/m²/s irradiance.Leaf material was harvested, flash frozen and ground in liquid nitrogen.Total RNA was extracted (Verwoerd et al. (1989) NAR 17, 2362) andNorthern blot analysis was carried out as described (Ward et al. (1991)The Plant Cell 3, 1085-1094) using radiolabelled probes specific foreach cellulase gene.

[0169] Transgenic lines with high levels of inducible transgeneexpression were allowed to flower and self-pollinate, producing T1seeds. Ten T1 seeds for each transgenic lines were germinated in soiland the resulting plants self-pollinated. T2 seeds from these plantswere germinated on T agar medium (Nitsch and Nitsch (1969) Science 163,85-87) containing 50 μg/ml hygromycin B to identify lines homozygous forthe selectable marker and linked transgene.

Example A11

[0170] Induction of cellulase expression in transgenic plants

[0171] Seeds of homozygous nuclear transformant lines are germinatedaseptically on T agar medium and incubated at 300 μmol/m²/s irradiancefor approximately 4-6 weeks. Alternatively, seeds are germinated in soiland grown in the greenhouse for approximately 2 months. Material oflines expressing cellulase genes under constitutive expression (CAMV 35Spromoter) is harvested and flash frozen in liquid nitrogen directly,while lines containing cellulase genes fused to the chemically induciblePR-1a promoter are first sprayed with either 1 mg/ml BTH or water,incubated for 1 to 7 days, and material harvested and flash frozen.

Example A12

[0172] Determination of cellulase content of transgenic plants

[0173] In order to determine the amount of cellulase present in thetissues of transgenic plants, chemiluminescent (Amersham) Western blotanalysis is performed according to the manufacturer's instructions andHarlow and Lane (1988) Antibodies: A laboratory manual, Cold SpringHarbor Laboratory, Cold Spring Harbor using antisera raised against theE1, E2 and E5 proteins and purified E1, E2 and E5 protein standards(provided by D. Wilson, Cornell University, Ithaca, N.Y.).

Example A13

[0174] Determination of cellulase activity in transgenic plants

[0175] Leaf material is harvested as described above and homogenized inPC buffer (50 mM phosphate, 12 mM citrate, pH 6.5). A standard curve (10nanomolar to 10 micromolar) is prepared by diluting appropriate amountsof 4-methylumbelliferone (MU, Sigma Cat. No. M1381) in PC buffer.Duplicate 100 μl aliquots of each standard and duplicate 50 μl aliquotsof each sample are distributed to separate wells of a 96-well microtiterplate. 50 μl of 2 mM 4-methylumbelliferyl-b-D-cellobiopyranoside (MUC,Sigma Cat. No. M6018) prepared in PC buffer is then added to each samplewell and the plate is sealed to prevent evaporation and incubated for 30minutes at 55° C. or at other temperatures ranging from 37° C to 65° C.The reaction is stopped by adding 100 μl of 0.15 M glycine/NaOH (pH10.3) and the MU fluorescence emission at 460 nm resulting fromcellulase activity is measured with a microplate fluorometer (excitationwavelength=355 nm).

[0176] B. Vacuole-Targeted Expression of Cellulases

Example B1

[0177] Preparation of a chimeric gene containing the T. fusca E5cellulase coding sequence fused to the tobacco PR-1a promoter

[0178] Plasmid pD374 containing the T. fusca E5 gene (see Example A3)was used as template for PCR with a left-to-right “top strand” primerextending from position 1,135 to 1,156 in the E5 gene relative to theendogenous ATG and comprising an additional NcoI site at its left end(primer VAC1: CAT GCC ATG GGT GAG GCC TCC GAG CTG TTC C) and aright-to-left “bottom strand” primer whose sequence was homologous tothe 21 last bp of the E5 gene and including 21 bp of a vacuolartargeting sequence derived from a tobacco chitinase gene (Shinshi et al.(1990) Plant Mol. Biol. 14, 357-368, Neuhaus et al. (1991) Proc. NatI.Acad. Sci. USA 88, 10362-10366), the stop codon of the same tobaccochitinase gene and a SacI restriction site (primer VAC2: TGC GAG CTC TTACAT AGT ATC GAC TAA AAG TCC GGA CTG GAG CTT GCT CCG CAC). AmpliTaq DNApolymerase was used for PCR according to the manufacturer'srecommendations for five cycles at 94° C. (30 s), 40° C. (60 s), and 72°C. (30 s) followed by 25 cycles at 94° C. (30 s), 55° C. (60 s) and 72°C. (30 s). This generated a product of 283-bp containing the 3′ end ofthe E5 gene fused to the vacuolar targeting sequence. The fragment wasgel purified, cleaved with NcoI and SacI and ligated into the NcoI andSacI sites of pJG203 to obtain pJGDE5.

[0179] Plasmid pD374 was then digested with NcoI and SacI, the 1.1 kblong fragment containing the 5′ end of the E5 gene including the signalsequence gel purified and ligated into the NcoI and SacI sites of pJGDE5to obtain pVACE5 containing the complete E5 gene with signal sequenceand vacuolar targeting sequence fused to a 903 bp long tobacco PR-1apromoter (FIG. 1).

[0180] Plasmid pVACE5 was digested with ApaI, XbaI and ScaI and theresulting 2.8 kb fragment containing the chimeric E5 gene was gelpurified and ligated into the ApaI and XbaI sites of pBHYGM to obtainpEGL115.

Example B2

[0181] Preparation of a chimeric gene containing the T. fusca E1cellulase coding sequence fused to the tobacco PR-1a promoter

[0182] A binary Agrobacterium transformation vector containing the T.fusca E1 cellulase coding sequence, its signal sequence, and a vacuolartargeting sequence fused to the tobacco PR-1a promoter is constructed asdescribed in Example B1 for the T. fusca E5 cellulase coding sequence.

Example B3

[0183] Preparation of a chimeric gene containing the T. fusca E2cellulase coding sequence fused to the tobacco PR-1 a promoter

[0184] A binary Agrobacterium transformation vector containing the T.fusca E2 cellulase coding sequence, its signal sequence, and a vacuolartargeting sequence fused to the tobacco PR-1 a promoter is constructedas described in Example B1 for the T. fusca E5 cellulase codingsequence.

Example B4

[0185] Preparation of a chimeric gene containing the T. fusca E5cellulase coding sequence fused to the CaMV 35S promoter

[0186] Plasmid pVACE5 was digested with NcoI and EcoICRI. The resulting1.6 kb fragment whose protruding NcoI ends had been previously filled-inwith Klenow DNA Polymerase was gel purified and ligated into thefilled-in EcoRI site of pCGN1761 to obtain p35SVACE5, containing the E5gene with signal sequence and vacuolar targeting sequence fused to theCaMV 35S promoter (FIG. 1). A 4.7 kb long fragment of p35SE5 containingthe chimeric E5 gene was inserted into the Xbal site of pBHYGM toconstruct pEGL315.

Example B5

[0187] Preparation of a chimeric gene containing the T. fusca E1cellulase coding sequence fused to the CaMV 35S promoter

[0188] A binary Agrobacterium transformation vector containing the T.fusca E1 cellulase coding sequence, its signal sequence, and a vacuolartargeting sequence fused the CaMV 35S promoter is constructed asdescribed in Example B4 for the T. fusca E5 cellulase coding sequence.

Example B6

[0189] Preparation of a chimeric gene containing the T. fusca E2cellulase coding sequence fused to the CaMV 35S promoter

[0190] A binary Agrobacterium transformation vector containing the T.fusca E2 cellulase coding sequence, its signal sequence, and a vacuolartargeting sequence fused to the CaMV 35S promoter is constructed asdescribed in Example B4 for the T. fusca E5 cellulase coding sequence.

Example B7

[0191] Transformation of tobacco leaf discs by A. tumefaciens

[0192] The binary vector constructs pEGL115 and pEGL315 were transformedinto A. tumefaciens strain GV3101 (Bechtold, N. et al. (1993), CR Acad.Sci. Paris, Sciences de la vie, 316:1194-1199) by electroporation(Dower, W. J. (1987), Plant Mol. Biol. Reporter 1:5). The same procedureis used for transformation of tobacco with other constructs containingchimeric cellulase genes.

[0193] Leaf discs of Nicotiana tabacum cv ‘Xanthi nc’ and of transgenicline “NahG” overexpressing a salicylate hydroxylase gene (Gaffney et al.(1993) Science 261: 754-756) were cocultivated with Agrobacterium clonescontaining the above mentioned constructs (Horsch et al. (1985), Science227: 1229-1231) and transformants were selected for resistance to 50μg/ml hygromycin B. Approximately 50 independent hygromycin lines (T0lines) for each construct were selected and rooted on hormone-freemedium.

Example B8

[0194] Transformation of maize

[0195] Maize transformation by particle bombardment of immature embryosis performed as described by Koziel et al. (Biotechnology 11, 194-200,1993).

Example B9

[0196] Transformation of wheat

[0197] Transformation of immature wheat embryos and immatureembryo-derived callus using particle bombardment is performed asdescribed by Vasil et. al. (Biotechnology 11: 1553-1558,1993) and Weekset. al. (Plant Physiology 102: 1077-1084, 1993).

Example B10

[0198] Selection of transgenic lines with inducible cellulase geneexpression

[0199] For each transgenic line duplicate leaf punches of approximately2-3 cm² were incubated for 2 days in 3 ml ofbenzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH, 5.6mg/10 ml) or sterile distilled water under ca. 300 μmol/m²/s irradiance.Leaf material was harvested, flash frozen and ground in liquid nitrogen.Total RNA was extracted (Verwoerd et al. (1989) NAR 17, 2362) andNorthern blot analysis was carried out as described (Ward et al. (1991)The Plant Cell 3, 1085-1094) using radiolabelled probes specific foreach cellulase gene.

[0200] Transgenic lines with high levels of inducible transgeneexpression were allowed to flower and self-pollinate, producing T1seeds. Ten T1 seeds for each transgenic lines were germinated in soiland the resulting plants self-pollinated. T2 seeds from these plantswere germinated on T agar medium (Nitsch and Nitsch (1969) Science 163,85-87) containing 50 μg/ml hygromycin B to identify lines homozygous forthe selectable marker and linked transgene.

Example B11

[0201] Induction of cellulase expression in transgenic plants

[0202] Seeds of homozygous nuclear transformant lines are germinatedaseptically on T agar medium and incubated at 300 μmol/m²/s irradiancefor approximately 4-6 weeks. Alternatively, seeds are germinated in soiland grown in the greenhouse for approximately 2 months. Material oflines expressing cellulase genes under constitutive expression (CaMV 35Spromoter) is harvested and flash frozen in liquid nitrogen directly,while lines containing cellulase genes fused to the chemically induciblePR-1a promoter are first sprayed with either 1 mg/ml BTH or water,incubated for 1 to 7 days, and material harvested and flash frozen.

Example B12

[0203] Determination of cellulase content of transgenic plants

[0204] In order to determine the amount of cellulase present in thetissues of transgenic plants, chemiluminescent (Amersham) Western blotanalysis is performed according to the manufacturer's instructions andHarlow and Lane (1988) Antibodies: A laboratory manual, Cold SpringHarbor Laboratory, Cold Spring Harbor using antisera raised against theE1, E2 and E5 proteins and purified E1, E2 and E5 protein standards(provided by D. Wilson, Cornell University, Ithaca, N.Y).

Example B13

[0205] Determination of cellulase activity in transgenic plants

[0206] 1. Fluorometric Assay

[0207] Leaf material is harvested as described above and homogenized inPC buffer (50 mM phosphate, 12 mM citrate, pH 6.5). A standard curve (10nanomolar to 10 micromolar) is prepared by diluting appropriate amountsof 4-methylumbelliferone (MU, Sigma Cat. No. M-1381) in PC buffer.Duplicate 100 μl aliquots of each standard and duplicate 50 μl aliquotsof each sample are distributed to separate wells of a 96-well microtiterplate. 50 μl of 2 mM 4-methylumbelliferyl-b-D-cellobiopyranoside (MUC,Sigma Cat. No. M6018), prepared in PC buffer is then added to each welland the plate is sealed to prevent evaporation and then incubated for 30minutes at the desired temperature (55_C.- 60_C. is optimal for T. fuscacellulases). The reaction is stopped by adding 100 μl of 0.15 Mglycine/NaOH (pH 10.3) and the fluorescence emission at 460 nm measuredwith a microplate fluorometer (excitation wavelength=355 nm). In orderto calculate the cellulase specific activity (pmoles MU/mgprotein/minute) the amount of protein in each extract is determinedusing a BCA assay (Pierce, Rockford, Ill.) according to themanufacturer's recommendations.

[0208] 2. CMCase Activity (according to Wilson (1988) Methods inEnzymology, 160: 314-315)

[0209] Leaf material is homogenized in 0.3 ml of 0.05 M potassiumphosphate buffer (pH 6.5) and is incubated with 0.1 ml ofcarboxymethylcellulose (CMC, Sigma , Cat. No. C-5678) for 15-60 minutesat the desired temperature (55_C.- 60_C. is optimal for T. fuscacellulases). After adding 0.75 ml of DNS reagent (200 g/l sodiumpotassium tartrate, 10 g/l dinitrosalicylic acid, 2 g/l phenol, 0.5 g/lsodium sulfite, 10 g/l sodium hydroxide) the samples are boiled for 15minutes. The samples are cooled down and the optical density is measuredat 600 nm. The amount of reducing sugars released from CMC is determinedusing a glucose standard curve and the cellulase activity is expressedin mmol glucose equivalent reducing sugar per minute. In order tocalculate the specific cellulase activity the amount of protein in eachextract is determined using a BCA assay (Pierce, Rockford, Ill.)according to the manufacturer's recommendations.

[0210] Alternatively, the cellulase activity on CMC is measured with aviscosity method as described by Durbin and Lewis (1988) Methods inEnzymology, 160: 314-315.

[0211] 3. Filter Paper Assay (according to Wilson (1988) Methods inEnzymology, 160: 314-315, thereby incorporated by reference)

[0212] Leaf material is homogenized in 0.05 M potassium phosphate buffer(pH 6.5) and the resulting extracts are added to a disk of filter paper(Whatman No. 1). After incubation for 4-24 hours at the desiredtemperature (55_C.- 60_C. is optimal for T. fusca cellulases), thereaction is stopped and reducing sugars content is determined.Alternatively, the cellulase activity on CMC is measured with aviscosity method as described by Durbin and Lewis (1988) Methods inEnzymology, 160: 314-315.

[0213] C. Expression of Cellulase Genes within the Tobacco Chloroplast

Example C1

[0214] Preparation of a chimeric gene containing the T. fusca E5cellulase coding sequence fused to a modified bacteriophage T7 gene 10promoter and terminator in tobacco plastid transformation vector pC8

[0215] Plasmid pCTE5 was digested with EcoRI, treated with Klenow DNApolymerase to fill in the recessed 3′ ends, digested with NcoI and theresulting 1.5 kb DNA fragment gel purified and ligated to a 7.5 kb NcoI(cohesive end)lXbal (filled in) DNA fragment from plastid transformationvector pC8 to create plasmid pC8E5 (FIG. 2). pC8 (Dr. Pal Maliga,Rutgers University, unpublished) is a derivative of plastidtransformation vector pPRV111A (Zoubenko, O. V., Allison, L. A., Svab,Z., and Maliga, P. (1994) Nucleic Acids Res 22, 3819-3824, hereinincorporated by reference in its entirety; GenBank accession numberU12812) that carries a bacterial aminoglycoside-3′-adenyltransferase(aadA) gene conferring spectinomycin resistance under control of theconstitutive tobacco plastid psbA gene promoter and psbA 5′ and 3′untranslated RNA (UTR) sequences. The 3′ end of the aadA cassette inpPRV111A is flanked by 1.8 kb of tobacco plastid DNA containing thecomplete trnV gene and a 5′ portion of the 16S rDNA gene while the 5′end is immediately adjacent to a multiple cloning site (EcoRI, SacI,KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI, HindIII) which is in turnflanked by the 1.2 kb of plastid DNA containing the ORF 70B gene and aportion of the rps 7/12 operon. These flanking homologous regions serveto target integration of the intervening heterologous DNA into theinverted repeat region of the tobacco plastid genome at nucleotidepositions 102,309 and 140,219 of the published Nicotiana tabacum plastidgenome sequence (Shinozaki, K. et al. (1986) EMBO J. 5, 2043-2049). pC8was obtained by cloning into the EcoRI and HindIII sites of the pPRV111Apolylinker a chimeric E. coli uidA gene encoding β-galacturonidase (GUS)controlled by the bacteriophage T7 gene 10 promoter and terminatorsequences derived from the pET21d expression vector (Novagen, Inc.,Madison, Wis.).

Example C2

[0216] Preparation of a modified tobacco plastid transformation vectorcontaining the T. fusca E5 cellulase coding sequence fused to a modifiedbacteriophage T7 gene 10 promoter and terminator engineered for reducedread-through transcription

[0217] Plasmid pC8 was digested with SpeI and NcoI and a 235 bp fragmentcontaining the T7 gene 10 promoter and a portion of the divergent psbAgene promoter and 5′ UTR was isolated by gel purification and clonedinto the NcoI and SpeI restriction sites of vector pGEM5Zf+ (Promega,Madison Wis.) to construct plasmid pPH118. pPH118 was digested with StuIand the 3210 bp vector fragment gel purified and religated to constructplasmid pPH119 which lacks the duplicated 10 bp sequence CGAGGCCTCG(StuI site underlined) that was found by sequence analysis to be presentin plasmid pC8. Elimination of the 10 bp Stul/StuI fragment in pPH119was verified by sequencing using universal M13 forward and reverseprimers.

[0218] In order to obtain a non-plastid DNA fragment to use as a spacerbetween the chimeric psbA/aadA selectable marker gene and the pET21d T7gene 10 promoter in pC8, yeast shuttle vector pRS305 (Sikorski, R. S.,and Hieter, P. (1989) Genetics 122, 19-27; GenBank accession numberU03437) was digested with EcoRI and HincII and a 256 bp fragment of theSaccharomyces cerevisiae LEU2 gene coding sequence isolated and gelpurified. Plasmid pPH119 was digested with EcoRI and DraIII and a 2645bp fragment isolated and gel purified. pPH119 was digested with EcoRI,treated with Klenow DNA polymerase to fill in the overhanging 3′terminus, digested with DraIII and a 569 bp fragment gel purified. Thethree fragments were ligated to create plasmid pPH120 in which the LEU2gene fragment is inserted between the divergent T7 gene 10 and psbApromoters of pPH119.

[0219] Plastid transformation vector pC+E5 (FIG. 2) was constructed bydigesting plasmid pPH120 with NcoI/EcoRI and gel purifying a 386 bpfragment, digesting plasmid pC8E5 with NcoI/HindIII and gel purifying a1595 bp fragment, digesting plasmid pC8 with HindIII/EcoRI and gelpurifying a 7287 bp fragment, and ligating the fragments in a 3-wayreaction.

Example C3

[0220] Construction of a plastid-targeted bacteriophage T7 RNApolymerase gene fused to the tobacco PR-1a promoter

[0221] A synthetic oligonucleotide linker comprising an NcoI restrictionsite and ATG start codon followed by the first seven plastid transitpeptide codons from the rbcS gene (encoding the small subunit ofribulose bisphosphate carboxylase) and endogenous PstI restriction site(top strand: 5′-CAT GGC TTC CTC AGT TCT TTC CTC TGC A-3′; bottom strand:5′-GAG GAA AGA ACT GAG GAA GC-3′), a 2.8 kb PstI/SacI DNA fragment ofpCGN4205 (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) containingthe bacteriophage T7 RNA polymerase gene (T7 Pol) fused in frame to the3′ portion of the rbcS gene transit peptide coding sequence, a 0.9 kbNcoI/KpnI DNA fragment of pCIB296 containing the tobacco PR-1a promoterwith an introduced NcoI restriction site at the start codon (Uknes etal. (1993) Plant Cell 5, 159 169) and 4.9 kb SfiI/KpnI and 6.6 kbSacI/SfiI fragments of binary Agrobacterium transformation vectorpSGCGC1 (a derivative of pGPTV-Hyg containing the polylinker from pGEM4(Promega, Madison Wis.) cloned into the SacI/HindIII sites) were ligatedto construct pPH110.

Example C4

[0222] Biolistic transformation of the tobacco plastid genome

[0223] Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ were germinated sevenper plate in a 1″ circular array on T agar medium and bombarded in situ12-14 days after sowing with 1 μm tungsten particles (M10, Biorad,Hercules, Calif.) coated with DNA from plasmids pC8E5 and pC+E5essentially as described (Svab, Z. and Maliga, P. (1993) PNAS 90, 913917). Bombarded seedlings were incubated on T medium for two days afterwhich leaves were excised and placed abaxial side up in bright light(350-500 μmol photons/m2/s) on plates of RMOP medium (Svab, Z.,Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526 8530) containing500 μg/ml spectinomycin dihydrochloride (Sigma, St. Louis, Mo.).Resistant shoots appearing underneath the bleached leaves three to eightweeks after bombardment were subcloned onto the same selective medium,allowed to form callus, and secondary shoots isolated and subcloned.Complete segregation of transformed plastid genome copies to ahomoplastidic state in independent subclones was assessed by standardtechniques of Southern blotting (Sambrook et al., (1989) MolecularCloning: A Laboratory Manual, Cold Springs Harbor Laboratory, ColdSpring Harbor). BamHI/EcoRI-digested total cellular DNA (Mettler, I. J.(1987) Plant Mol Biol Reporter 5, 346-349) was separated on 1%Trisborate (TBE) agarose gels, transferred to nylon membranes (Amersham)and probed with ³²P-labeled random primed DNA sequences corresponding toa 0.7 kb BamHI/HindIII fragment from pC8 containing a portion of therps7/12 plastid targeting sequence. Homoplastidic shoots were rootedaseptically on spectinomycin-containing MS/IBA medium (McBride, K. E. etal., (1994) PNAS 91, 7301-7305) and transferred to the greenhouse.

Example C5

[0224] Introduction of the chimeric PR-1a/T7 Pol gene into the tobacconuclear genome by Agrobacterium-mediated leaf disc transformation

[0225] Hygromycin resistant NT-pPH110 tobacco plants were regenerated asdescribed from shoots obtained following cocultivation of leaf disks ofN. tabacum ‘Xanthi’ and “NahG” with GV3101 Agrobacterium carrying thepPH110 binary vector. For each transgenic line duplicate leaf punches ofapproximately 2-3 cm² were incubated for 2 days in 3 ml of BTH (5.6mg/10 ml) or sterile distilled water under ca. 300 μmol/m²/s irradiance.Leaf material was harvested, flash frozen and ground in liquid nitrogen.Total RNA was extracted (Verwoerd et al. (1989) NAR 17, 2362) andNorthern blot analysis was carried out as described (Ward et al. (1991)The Plant Cell 3, 1085-1094) using a radiolabelled T7 RNA polymerasegene probe. Plants of nineteen NT-110X (Xanthi genetic background) andseven NT-110N (NahG genetic background) T1 lines showing a range of T7Pol expression were transferred to the greenhouse and self pollinated.Progeny segregating 3:1 for the linked hygromycin resistance marker wereselfed and homozygous T2 lines selected.

Example C6

[0226] Induction of cellulase expression in plastids of transgenicplants

[0227] Homozygous NT-110X and NT-110N plants containing the PR-1a-T7 RNAPol construct were used to pollinate homoplastidic Nt_pC8E5 and Nt_pC+E5plastid transformant lines carrying the maternally inherited pC8E5 andpC+E5 cellulase constructs. The Nt_pC+E5×NT-110X or NT_(—)110N, andNt_pC8E5×NT-110X or NT_(—)110N F1 progeny (which were heterozygous forthe PR-1/T7 polymerase nuclear expression cassette and homoplastidic forthe T7/cellulase plastid expression cassette) were germinated on soil.Upon reaching a height of 20-40 cm, the plants were sprayed with theinducer compound BTH to elicit T7 Pol-regulated expression of the E5cellulase gene that is localized to the plastids. Plant material washarvested just prior to induction and at 8 hours and 1, 2, 3, 7, and 14or 28 days following induction and flash frozen as described above.

Example C7

[0228] Determination of E5 cellulase mRNA content of transgenic plants

[0229] Total RNA was extracted from frozen tissue of BTH and wettablepowder-sprayed control and PR-1a/7 polymerase×plastid T7/cellulaseplants, and Northern blot analysis on 5 μg RNA samples was carried outas described (Ward et al. (1991) The Plant Cell 3, 1085-1094) using as aprobe a radiolabelled DNA fragment containing the E5 cellulase codingsequence. Relative E5 cellulase MRNA accumulation at each time point wasassessed by quantifying the radioactivity in bands hybridizing with theradiolabelled E5 cellulase probe in order to determine time courses offold mRNA induction. The transgenic plant material of example C6 showssignificant cellulase mRNA accumulation in this assay followinginduction, peaking at 14 days after induction. Prior to induction, nocellulase MRNA is detected.

Example C8

[0230] Determination of cellulase content of transgenic plants

[0231] In order to determine the amount of cellulase present in thetissues of transgenic plants, chemiluminescent (Amersham) Western blotanalysis is performed according to the manufacturer's instructions andHarlow and Lane (1988) Antibodies: A laboratory manual, Cold SpringHarbor Laboratory, Cold Spring Harbor using antisera raised against theE5 protein and purified E5 protein standards (provided by D. Wilson,Cornell University, Ithaca, N.Y). The transgenic plant material ofexample C6 shows significant cellulase expression and accumulation inthis assay following induction (ca. 0.3% of total soluble protein at 14days after induction; no detectible protein prior to induction).

Example C9

[0232] Determination of cellulase activity in transgenic plants

[0233] Leaf material is harvested as described above and homogenized inPC buffer (50 mM phosphate, 12 mM citrate, pH 6.5). A standard curve (10nanomolar to 10 micromolar) is prepared by diluting appropriate amountsof 4-methylumbelliferone (MU, Sigma Cat. No. M1381) in PC buffer.Duplicate 100 μl aliquots of each standard and duplicate 50 μl aliquotsof each sample are distributed to separate wells of a 96-well microtiterplate. 50 μl of 2 mM 4-methylumbelliferyl-b-D-cellobiopyranoside (MUC,Sigma Cat. No. M6018) prepared in PC buffer is then added to each samplewell and the plate is sealed to prevent evaporation and incubated for 30minutes at 55° C. or at other temperatures ranging from 37° C. to 65° C.The reaction is stopped by adding 100 μl of 0.15 M glycine/NaOH (pH10.3) and the fluorescence emission at 460 nm measured with a microplatefluorometer (excitation wavelength=355 nm).

Example C10

[0234] Induction of GUS expression in plastids of transgenic plants

[0235] The N. tabacum ‘Xanthi’ plastid transformant line 4276P describedby McBride et al. ((1994) PNAS 91: 7301-7305) was pollinated byhomozygous NT-110X6b-5 plants containing the PR-1a/T7 RNA polymerase.4276P differs from pC8 only with respect to (a) the promoter used toexpress the aadA selectable marker (which has the 16S ribosomal RNA genepromoter rather than the psbA gene promoter used in pC8), (b) thepresence of a psbA gene 3′ untranslated region between the GUS gene andthe T7 terminator, and (c) the absence of a lac operator and duplicatedStul restriction site sequence in the T7 promoter. F1 plants from thiscross heterozygous for the PR-1a/T7 polymerase nuclear expressioncassette and homoplastidic for the T7/GUS plastid expression cassettewere germinated in soil. Upon reaching a height of 20 to 40 cm theplants were sprayed with either an inert wettable powder suspension or aformulation of the inducer compound BTH with wettable powder. Controluntransforned N. tabacum ‘Xanthi’, NT-110X6b-5, and 4276P plantsgerminated in soil at the same time were sprayed in a similar manner.Plant material (one leaf from each of three independent plants of eachgenotype) was harvested just prior to spraying and at 8 hours and 1, 2,3, 7, and 28 days following spraying, and flash frozen as describedabove.

Example C11

[0236] Determination of GUS MRNA content of transgenic plants

[0237] Total RNA was extracted from frozen tissue of BTH and wettablepowder-sprayed control and PR-1a/T7 polymerase×plastid T7/GUS plants,and Northern blot analysis on 5 μg RNA samples was carried out asdescribed (Ward et al. (1991) The Plant Cell 3, 1085-1094) using as aprobe a 500 bp radiolabelled 5′ fragment of the GUS gene. GUS mRNAaccumulation at each time point was assessed by quantifying theradioactivity in bands hybridizing with the radiolabelled GUS probe, aswell as by scanning the ethidium-bromide fluorescence present in theprominent RNA band which became visible starting at the 3 day post-spraytime point and which was observed to co-migrate with the hybridizing GUSRNA band on Northern blots. Chemically inducible GUS RNA in the plastidwas observed to reach a peak level of 14% of total ethidium-stainableRNA (this includes all RNA species present in the plant, including thenon-protein coding ribosomal RNA which makes up a majority of thestainable plant RNA) between 7 and 28 days after induction with BTH (seeTable 1) and is much higher (over 1000-fold) than the peak chemicallyinducible GUS mRNA accumulation for nuclear PR- I a/GUS transformants.

Example C12

[0238] Determination of GUS protein content of transgenic plants

[0239] In order to determine the amount of GUS present in the tissues oftransgenic plants, chemiluminescent (Amersham) Western blot analysis wasperformed according to the manufacturer's instructions and Harlow andLane (1988) Antibodies: A laboratory manual, Cold Spring HarborLaboratory, Cold Spring Harbor, using GUS antisera purchased fromMolecular Probes and purified GUS protein standards (Sigma). Proteinsfrom frozen, ground leaf material harvested as above were solubilized byextraction in 50 mM Tris pH 8.0, 1 mM EDTA, 1 mM PTT, 1 mM AEBSP, and 1mM DTT and 5 to 25 μg protein run on each lane of 10% polyacrylamidegels. GUS protein accumulation in the plastid transformed plants issustained over 7-28 days and beyond, and is extraordinarily high (muchhigher than peak GUS accumulation for nuclear PR-1a/GUS), exceeding 20%of of total protein by 28 days. By comparison, GUS protein accumulationin the nuclear PR1a/GUS transformants peaks somewhat earlier (about 3days from induction, rather than 7-28 days) and the protein accumulationis not sustained, but declines to the limits of detection by 28 days.

Example C13

[0240] Determination of GUS activity in transgenic plants

[0241] Frozen leaf tissue was ground in a mortar with a pestle in thepresence of liquid nitrogen to produce a fine powder. Leaf extracts wereprepared in GUS extraction buffer (50 mM sodium phosphate pH7.0, 0.1%Triton-X 100, 0.1% sarkosyl, 10 mM beta-mercaptoethanol) as described byJefferson, R. A. et al. (1986), PNAS USA 83, 8447-8451. The reactionswere carried out in the wells of opaque microtiter plates by mixing 10ul of extract with 65 ul of GUS assay buffer (50 mM sodium phosphate pH7.0, 10 mM EDTA, 0.1% Triton X-100, 10 mM beta-mercaptoethanol)containing 4-methyl umbelliferyl glucuronide (MU) at a finalconcentration of 2 mM in a total volume of 75 ul. The plate wasincubated at 37° C. for 30 minutes and the reaction was stopped by theaddition of 225 ul of 0.2 M sodium carbonate. The concentration offluorescent indicator released was determined by reading the plate on aFlow Labs Fluoroskan II ELISA plate reader. Duplicate fluorescencevalues for each sample were averaged, and background fluorescence(reaction without MUG) was subtracted to obtain the concentration of MUfor each sample. The amount of protein in each extract was determinedusing the bicinchoninic acid technique (BCA, Pierce Biochemicals)according to the manufacturer's recommendations except that proteinextracts were pretreated with iodacetamide (Sigma) to eliminatebackground signal caused by the reductant (beta-mercaptoethanol) presentin the extraction buffer. The specific activity was determined for eachsample and was expressed in pmoles MU/mg protein/minute. For each tissuesample assayed from a particular time point following BTH application,the specific activity of the BTH-induced sample was divided by thespecific activity of the pre-BTH treatment control sample, thus yieldingthe induction of GUS expression. (See Table I) TABLE I pPH110X6b ×4276P: Induction of GUS RNA and GUS Activity by Spraying with BTH GUSactivity (pmol Fold Induction % Total Fold Induction Days + BTHMU/mg/min) (GUS activity) RNA (GUS RNA) 0.0 598 1 <0.013 1 0.3 434 0.7<0.013 24 1.0 14,516 24 0.108 959 2.0 230,031 380 0.873 1,897 3.0456,486 749 2.663 2,396 7.0 2,424,725 3,999 7.745 2,875 28.0 1,922,4663,106 24.596 3,392

[0242]

1 19 13 base pairs nucleic acid single linear other nucleic acid /desc =“Consensus tranlation 1 GTCGACCATG GTC 13 12 base pairs nucleic acidsingle linear other nucleic acid /desc = ”Consensus translation 2TAAACAATGG CT 12 22 base pairs nucleic acid single linear other nucleicacid /desc = “Molecular adaptor used to 3 AATTCTAAAG CATGCCGATC GG 22 21base pairs nucleic acid single linear other nucleic acid /desc =”Molecular adaptor used to 4 AATTCCGATC GGCATGCTTT A 21 22 base pairsnucleic acid single linear other nucleic acid /desc = “Molecular adaptorused in 5 AATTCTAAAC CATGGCGATC GG 22 21 base pairs nucleic acid singlelinear other nucleic acid /desc = ”Molecular adaptor used in 6AATTCCGATC GCCATGGTTT A 21 15 base pairs nucleic acid single linearother nucleic acid /desc = “Molecular adaptor sequence 7 CCAGCTGGAATTCCG 15 19 base pairs nucleic acid single linear other nucleic acid/desc = ”Molecular adaptor sequence 8 CGGAATTCCA GCTGGCATG 19 30 basepairs nucleic acid single linear other nucleic acid /desc = “Primer E11”9 GCGCCCATGG ACGAAGTCAA CCAGATTCGC 30 24 base pairs nucleic acid singlelinear other nucleic acid /desc = “Primer E12” 10 CCAGTCGACG TTGGAGGTGAAGAC 24 31 base pairs nucleic acid single linear other nucleic acid/desc = “Primer E21” 11 GCGCGCCATG GCCAATGATT CTCCGTTCTA C 31 24 basepairs nucleic acid single linear other nucleic acid /desc = “Primer E22”12 GGGACGGTTC TTCAGTCCGG CAGC 24 29 base pairs nucleic acid singlelinear other nucleic acid /desc = “Primer E51” 13 CGCCCATGGC CGGTCTCACCGCCACAGTC 29 25 base pairs nucleic acid single linear other nucleic acid/desc = “Primer E52” 14 GACGACCTCC CACTGGGAGA CGGTG 25 31 base pairsnucleic acid single linear other nucleic acid /desc = “Primer VAC1” 15CATGCCATGG GTGAGGCCTC CGAGCTGTTC C 31 54 base pairs nucleic acid singlelinear other nucleic acid /desc = “Primer VAC2” 16 TGCGAGCTCT TACATAGTATCGACTAAAAG TCCGGACTGG AGCTTGCTCC GCAC 54 10 base pairs nucleic acidsingle linear other nucleic acid /desc = “Sequence present in plasmid 17CGAGGCCTCG 10 28 base pairs nucleic acid single linear other nucleicacid /desc = ”Top strand of 18 CATGGCTTCC TCAGTTCTTT CCTCTGCA 28 20 basepairs nucleic acid single linear other nucleic acid /desc = “Bottomstrand of 19 GAGGAAAGAA CTGAGGAAGC 20

What is claimed is:
 1. A plant comprising (a) a heterologous nuclearexpression cassette comprising an inducible promoter operably linked toa DNA sequence coding for a transactivator, and (b) a heterologousplastid expression cassette comprising a transactivator-mediatedpromoter regulated by said transactivator and operably linked to one ormore DNA sequences of interest.
 2. The plant according to claim 1wherein the inducible promoter is a wound-inducible orchemically-inducible promoter.
 3. The plant according to claim 2 whereinthe inducible promoter is the tobacco PR-1a promoter.
 4. The plantaccording to any of claims 1, 2 or 3 wherein the transactivator is T7RNA polymerase and the transactivator-mediated promoter is the T7promoter.
 5. The plant according to any of claims 1 through 4 wherein atleast one of the DNA sequences of interest in the heterologous plastidexpression cassette codes for a cellulose-degrading enzyme.
 6. A plantwhich expresses a cellulose degrading enzyme.
 7. The plant of claim 6comprising a heterologous DNA sequence coding for a cellulose degradingenzyme stably integrated into its nuclear or plastid DNA and undercontrol of an inducible promoter.
 8. The plant of claim 7 wherein theinducible promoter is a wound-inducible or chemically-induciblepromoter.
 9. The plant according to claim 8 wherein the induciblepromoter is PR-1.
 10. A package of seed of a plant according to any ofclaims 1 through 9.